15-PGDH in colon cancer

ABSTRACT

The disclosure provides, among other things, a method of decreasing resistance to the chemopreventive properties of non-steroidal anti-inflammatory agents, e.g., celecoxib, particularly in the prevention of cancer, e.g., colon cancer, by increasing the levels or activity of 15-hydroxyprostaglandin dehydrogenase (15-PGDH). The disclosure also provides a method of identifying compounds that upregulate or reactivate 15-PGDH. The disclosure also provides a method of identifying an individual suitable for treatment with a non-steroidal anti-inflammatory agent in the treatment or prevention of colon cancer.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/774,641(now U.S. Pat. No. 8,497,084), filed May 5, 2010, which claims thebenefit of priority to U.S. Provisional Application No. 61/215,505,filed May 5, 2009. The specification of the foregoing applications areincorporated herein by reference in their entirety.

BACKGROUND

Colon cancer is the second leading cause of cancer-related death in theUnited States (Markowitz, S. D. (2007) N Engl J Med 356, 2195-8). Anestimated 135,000 new cases of colon cancer occur each year. Althoughmany people die of colon cancer, early stage colon cancers are oftentreatable by surgical removal (resection) of the affected tissue.Surgical treatment can be combined with chemotherapeutic agents toachieve an even higher survival rate in certain colon cancers. However,the survival rate drops to 5% or less over five years in patients withmetastatic (late stage) colon cancer.

Additionally, systemic therapies in combination with chemotherapies havebeen developed for the treatment of colon cancer. However, no therapieshave exhibited sufficient anti-tumor activity to prolong the survival ofcolon cancer patients with metastatic disease with any degree ofreliability. Strategies for preventing colon cancer have focused onpreventing development of colonic adenomas, the premalignant tumors thatare the precursors of invasive colon cancers (Markowitz, S. D. (2007) NEngl J Med 356, 2195-8). For example, pharmacologic approaches havetargeted the inhibition of COX-2, an enzyme that mediates conversion ofarachidonic acid to bioactive prostaglandins, and whose expression ismarkedly increased in colon cancers (Markowitz, S. D. (2007) N Engl JMed 356, 2195-8; Cha, Y. I. & DuBois, R. N. (2007) Annu Rev Med 58,239-52) Inhibitors of COX-2 such as nonsteroidal anti-inflammatory drugs(NSAID) have been shown to decrease colon adenoma development inindividuals with Familial Adenomatous Polyposis (Steinbach, G. et al.(2000) N Engl J Med 342, 1946-52). For example, in individuals withnon-familial sporadic colon adenomas, celecoxib reduces by 33-45% therisk of developing future adenomas, and by 57-64% the risk of developingadenomas with advanced histology (Arber, N. et al., (2006) N Engl J Med355, 885-95; Bertagnolli, M. M. et al., (2006) N Engl J Med 355,873-84). However, a significant proportion of individuals demonstrateresistance to the colon tumor prevention activity of NSAIDs, and themolecular mechanism underlying this resistance is largely unknown.Accordingly, a need still exists to develop methods for the successfultreatment of colorectal carcinoma.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of identifyingsubjects who would be responsive to non-steroidal inflammatory drug(NSAID) therapy, comprising: (i) obtaining a biological sample from saidsubject; and (ii) measuring 15-PGDH levels or 15-PGDH activity, whereinnormal or increased level or activity of 15-PGDH indicates that thesubject would be responsive to NSAID therapy.

In certain embodiments, the 15-PGDH level or 15-PGDH activity iscompared to a reference level or activity of 15-PGDH. In one embodiment,the reference level or activity of 15-PGDH is measured from a healthysubject, or a subject known to be responsive to NSAID therapy. In oneembodiment, the NSAID therapy prevents or treats colon neoplasia, e.g.,colon adenoma or colon cancer. In other embodiments, the NSAID therapytreats pain disorders, inflammatory disorders, and immunologicdisorders.

In some embodiments, said NSAID is celecoxib or aspirin. In oneembodiment, the sample is a colonic tissue. In some embodiments, thesample is a bodily fluid selected from blood, serum, plasma, ablood-derived fraction, stool, urine, and a colonic effluent. In otherembodiments, the blood-derived fraction comprises peripheral bloodleukocytes.

In certain embodiments, the level measured in the foregoing method is aprotein, mRNA, or cDNA level of 15-PGDH.

In another aspect, the present invention provides a method ofdetermining whether a subject is predisposed to developing resistance toan NSAID therapy, comprising: (i) obtaining a biological sample fromsaid subject; and (ii) measuring 15-PGDH levels or 15-PGDH activity;wherein a reduced level or activity of 15-PGDH in the subject relativeto a reference sample indicates that the subject is predisposed todeveloping resistance in the NSAID therapy.

In any of the foregoing aspects and embodiments, the method furthercomprises (i) obtaining a reference biological sample from a normalsubject; (ii) measuring levels of 15-PGDH or its activity; and (iii)comparing said levels or activity in the sample from the subject tolevels or activity in a normal sample. In certain embodiments, the NSAIDtherapy prevents or treats colon neoplasia, e.g., colon adenoma or coloncancer. In other embodiments, the NSAID therapy treats pain disorders,inflammatory disorders, and immunologic disorders.

In some embodiments, the NSAID is celecoxib or aspirin. In otherembodiments, the sample comprises a colonic tissue. In otherembodiments, the sample is a bodily fluid selected from the groupconsisting of: blood, serum, plasma, a blood-derived fraction, stool,urine, and a colonic effluent. Exemplary blood-derived fraction includesperipheral blood leukocytes.

In some embodiments, the level measured in the foregoing method isselected from a protein, mRNA, or cDNA of 15-PGDH. In other aspects, thepresent invention provides a method of treating an NSAID-responsivecondition comprising administering an effective amount of an NSAID andan agent that upregulates 15-PGDH. In certain embodiments, the agent isnot a Prox-1 suppressor.

In certain embodiments, the NSAID-responsive condition is prevention ortreatment of colon neoplasia, e.g., colon cancer or colon adenoma. Inother embodiments, the NSAID-responsive condition is selected from thegroup consisting of: pain disorders, inflammatory disorders, orimmunologic disorders.

In some embodiments, the NSAID is celecoxib or aspirin. In certainembodiments, the agent is selected from the group consisting of: a smallmolecule, a polypeptide, nucleic acid, aptamers, or antibody. Exemplarysmall molecules include erlotinib or butyrate. In one embodiment, thenucleic acid is an siRNA or antisense. In certain embodiments, the siRNAinhibits the expression of beta-catenin transcription factor. In otherembodiments, the agent directly or indirectly upregulates 15-PGDH levelsor activity. In another aspect, the present invention provides a methodof decreasing NSAID resistance in a patient, comprising administering:(i) an effective amount of a compound that increases 15-PGDH levels or15-PGDH activity; or (ii) an effective amount of 15-PGDH protein, cDNA,or an active fragment thereof. In certain embodiments, a compound thatincreases 15-PGDH levels is not a Prox-1 suppressor.

In certain embodiments, the levels include protein, mRNA, or cDNA levelof 15-PGDH. In one embodiment, the compound is selected from the groupconsisting of: a small molecule, a polypeptide, a nucleic acid, anaptamer, or an antibody. Exemplary nucleic acids include an siRNA orantisense. In one embodiment, the siRNA inhibits the expression ofbeta-catenin transcription factor. In certain embodiments, the compounddirectly or indirectly upregulates 15-PGDH levels or activity.

In other aspects, the present invention provides a method of screeningfor test agents that would decrease non-steroidal inflammatory drug(NSAID) resistance, comprising: contacting cells with said test agent;and measuring 15-PGDH levels or 15-PGDH activity, wherein an increase in15-PGDH level or activity indicates that said agent is a candidate agentthat decreases NSAID resistance.

In some embodiments, the 15-PGDH level is compared to a reference cell.

In certain embodiments, the test agent is selected from the groupconsisting of: a small molecule, a polypeptide, nucleic acid, aptamers,and antibody. In exemplary embodiments, the nucleic acid is an siRNA orantisense. In one embodiment, the test agent directly or indirectlyupregulates 15-PGDH levels or activity. In one aspect, the presentinvention provides a method of identifying a subject who is at risk fordeveloping colon neoplasia, comprising: (i) obtaining a colon tissuesample from said subject; and (ii) measuring 15-PGDH levels or 15-PGDHactivity, wherein a level or activity of 15-PGDH that is less than 50%of the level or activity of 15-PGDH from a healthy population indicatesthat the subject is at risk for developing colon neoplasia. In certainembodiments, the level or activity of 15-PGDH from the subject sample isless than 90%, 80%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, or 5% of the level or activity of 15-PGDH determined froma healthy population. As described herein, the level of 15-PGDH is avalue of an amount of 15-PGDH protein or nucleic acid measured as aquantified unit. In certain embodiments, the activity of 15-PGDH is ameasured unit of enzymatic activity of 15-PGDH on a known substrate,e.g., PGE₂. In other embodiments, such levels are average valuesmeasured from a pool of a healthy population.

In some embodiments, the biological sample is selected from the groupconsisting of whole blood or a fraction thereof. In other embodiments,said biological sample is selected from the group consisting of urine orstool samples. In certain embodiments, the biological sample is a bloodsample. In certain embodiments, the sample is a non-neoplastic sample.In one embodiment, the blood sample is fractionated to obtain bloodserum and/or blood plasma. In other embodiments, the biological sampleis colonic tissue. In one embodiment, said biological sample is enrichedfor 15-PGDH. In some embodiments, the level is protein, mRNA or cDNAlevel of 15-PGDH.

In one embodiment, the 15-PGDH level is detected by an assay. In oneexemplary method, the assay employs an antibody. In one embodiment, theantibody is detectably labeled, e.g., an enzyme, a fluorescentsubstance, a chemiluminescent substance, a chromophore, a radioactiveisotope and a complexing agent. In other embodiments, the assay isselected from an immunoprecipitation assay, a Western blot, aradioimmunoassays, an enzyme-linked immunosorbent assay (ELISA), andPCR.

In one aspect, the present invention provides a kit for detecting colonneoplasia in a biological sample, comprising: a) an antibody whichinteracts with an epitope of 15-PGDH; and b) instructions for use. Inone embodiment, said antibody is detectably labeled, e.g., an enzyme, afluorescent substance, a chemiluminescent substance, a chromophore, aradioactive isotope and a complexing agent.

In another aspect, the present invention provides a kit for detectingcolon neoplasia in a biological sample, comprising: a) a primer capableof amplifying 15-PGDH nucleic acid; and b) instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Celecoxib resistance in 15-PGDH knockout mice. (A) AOM inducedcolon tumor development in 15-PGDH +/+ FVB mice untreated (−) (n=16) ortreated (+) (n=12) with celecoxib, versus FVB 15-PGDH −/− mice untreated(n=13) or treated (n=17) with celecoxib. P-values represent comparisonsof tumor numbers between groups, with * indicating statisticallysignificant values. Error bars designate standard error of the mean. (B)AOM induced development of large colon tumors (diameter>1 mm) in thesame mice cohorts graphed in panel A). (C) Gross morphology ofrepresentative colons from AOM treated 15-PGDH+/+ and −/− miceadministered celecoxib containing (+) or celecoxib free (−) diets.Arrows designate colon tumors. (D) Colonic mucosal PGE₂ levels (ng/mgprotein) in 15-PGDH +/+ FVB mice untreated (−) (n=10) or treated (+)(n=9) with celecoxib, versus FVB 15-PGDH −/− mice untreated (n=12) ortreated (n=11) with celecoxib.

FIG. 2: Celecoxib and 15-PGDH tissue levels in murine colonic mucosa.(A) Tissue levels of celecoxib were determined by mass spectrometry intissue homogenates of colonic mucosa obtained from 15-PGDH wild-type(+/+) (n=20) or knockout (−/−) (n=26) mice administered two weeks of acelecoxib supplemented diet. Error bars designate standard error of themean. Mice cohorts correspond to those of FIG. 1D. (B) Western analysisof 15-PGDH expression determined in colon mucosa from 3 sets of micereceiving two weeks of a control (−) or two weeks of acelecoxib-containing (+) diet. Actin protein levels serve as a loadingcontrol. FIG. 3: Celecoxib resistance in humans with low levels of15-PGDH. Shown on the X-axis are pre-treatment 15-PGDH transcript levelsmeasured by real-time PCR in RNA from rectal mucosal biopsies of 16individuals enrolled in the Adenoma Prevention with Celecoxib trial(Bertagnolli, M. M. et al, (2006) N Engl J Med 355, 873-84). Bar heightson the Y-axis indicate number of recurrent adenomas detected in eachindividual at the completion of 36 months of celecoxib treatment, withblue bars denoting individuals with recurrent disease, and withindividuals with zero recurrences indicated by minimal black bars.Median level of 15-PGDH is denoted by the dashed line (and indicated byan arrow), and the mean level is denoted by the dashed line (notindicated by arrow).

FIG. 4: Established colon cancer cell line for screening 15-PGDHmodulators. By homologous recombination, a ranilla luciferase cassettewas inserted in-frame into the silent PGDH locus in the LS-174 T cells.The figure illustrates the induction of the 15-PGDH-ranilla luciferasefusion protein as measured by luciferase activity upon treatment with abeta-catenin siRNA (denoted S4). Untransfected cells (UTX) and cellstransfected with a control scrambled siRNA (“scramble”) are also shownfor comparison.

DETAILED DESCRIPTION

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “neoplasm” refers to any abnormal mass of cells or tissue as aresult of neoplasia. The neoplasm may be benign, potentially malignant(precancerous), or malignant (cancerous). An adenoma is an example of aneoplasm.

The terms “adenoma”, “colon adenoma” and “polyp” are used herein todescribe any precancerous neoplasm of the colon.

The term “colon” as used herein is intended to encompass the right colon(including the cecum), the transverse colon, the left colon and therectum.

The terms “colorectal cancer” and “colon cancer” are usedinterchangeably herein to refer to any cancerous neoplasia of the colon(including the rectum, as defined above).

As used herein, the phrase “gene expression” or “protein expression”includes any information pertaining to the amount of gene transcript orprotein present in a sample, as well as information about the rate atwhich genes or proteins are produced or are accumulating or beingdegraded (eg. reporter gene data, data from nuclear runoff experiments,pulse-chase data etc.). Certain kinds of data might be viewed asrelating to both gene and protein expression. For example, proteinlevels in a cell are reflective of the level of protein as well as thelevel of transcription, and such data is intended to be included by thephrase “gene or protein expression information”. Such information may begiven in the form of amounts per cell, amounts relative to a controlgene or protein, in unitless measures, etc.; the term “information” isnot to be limited to any particular means of representation and isintended to mean any representation that provides relevant information.The term “expression levels” refers to a quantity reflected in orderivable from the gene or protein expression data, whether the data isdirected to gene transcript accumulation or protein accumulation orprotein synthesis rates, etc.

The term “detection” or “measuring” is used herein to refer to anyprocess of observing a marker, in a biological sample, whether or notthe marker is actually detected. In other words, the act of probing asample for a marker is a “detection” or “measurement” even if the markeris determined to be not present or below the level of sensitivity.Detection or measurement may be a quantitative, semi-quantitative ornon-quantitative observation.

The terms “healthy”, “normal” and “non-neoplastic” are usedinterchangeably herein to refer to a subject or particular cell ortissue that is devoid (at least to the limit of detection) of a diseasecondition, such as a neoplasia, that is associated with colon cancer.These terms are often used herein in reference to tissues and cells ofthe colon. Thus, for the purposes of this application, a patient withsevere heart disease but lacking a colorectal cancer-associated diseasewould be termed “healthy”.

As used herein, a “reference sample” is a sample derived from a healthy,normal, or non-neoplastic subject.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”.

As used herein, the term “nucleic acid” refers to polynucleotides suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include analogs of eitherRNA or DNA made from nucleotide analogs, and, as applicable to theembodiment being described, single-stranded (such as sense or antisense)and double-stranded polynucleotides. In some embodiments, “nucleic acid”refers to inhibitory nucleic acids. Some categories of inhibitorynucleic acid compounds include antisense nucleic acids, RNAi constructs,and catalytic nucleic acid constructs. Such categories of nucleic acidsare well-known in the art.

“Treating” a condition or disease refers to curing as well asameliorating at least one symptom of the condition or disease.

The term “prophylactic” or “therapeutic” treatment is art-recognized andrefers to administration of a drug to a host. The terms “prophylactic”and “therapeutic” is used interchangeably with “prevent”/“prevention”and “treat”/treatment, respectively. If it is administered prior toclinical manifestation of the unwanted condition (e.g., disease or otherunwanted state of the host animal) then the treatment is prophylactic,i.e., it protects the host against developing the unwanted condition,whereas if administered after manifestation of the unwanted condition,the treatment is therapeutic (i.e., it is intended to diminish,ameliorate or maintain the existing unwanted condition or side effectstherefrom).

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, or any other medical condition, is well understood in the art,and includes administration of a composition which reduces the frequencyof, or delays the onset of, symptoms of a medical condition in a subjectrelative to a subject which does not receive the composition. Thus,prevention of cancer includes, for example, reducing the number ofdetectable cancerous growths in a population of patients receiving aprophylactic treatment relative to an untreated control population,and/or delaying the appearance of detectable cancerous growths in atreated population versus an untreated control population, e.g., by astatistically and/or clinically significant amount. Prevention of aninfection includes, for example, reducing the number of diagnoses of theinfection in a treated population versus an untreated controlpopulation, and/or delaying the onset of symptoms of the infection in atreated population versus an untreated control population. Prevention ofpain includes, for example, reducing the magnitude of, or alternativelydelaying, pain sensations experienced by subjects in a treatedpopulation versus an untreated control population.

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or”, unless context clearly indicates otherwise.

The terms “polypeptide” and “protein” are used interchangeably herein.

The term “purified protein” refers to a preparation of a protein orproteins which are preferably isolated from, or otherwise substantiallyfree of, other proteins normally associated with the protein(s) in acell or cell lysate. The term “substantially free of other cellularproteins” (also referred to herein as “substantially free of othercontaminating proteins”) is defined as encompassing individualpreparations of each of the component proteins comprising less than 20%(by dry weight) contaminating protein, and preferably comprises lessthan 5% contaminating protein. Functional forms of each of the componentproteins can be prepared as purified preparations by using a cloned geneas described in the attached examples. By “purified”, it is meant, whenreferring to component protein preparations used to generate areconstituted protein mixture, that the indicated molecule is present inthe substantial absence of other biological macromolecules, such asother proteins (particularly other proteins which may substantiallymask, diminish, confuse or alter the characteristics of the componentproteins either as purified preparations or in their function in thesubject reconstituted mixture). The term “purified” as used hereinpreferably means at least 80% by dry weight, more preferably in therange of 85% by weight, more preferably 95-99% by weight, and mostpreferably at least 99.8% by weight, of biological macromolecules of thesame type present (but water, buffers, and other small molecules,especially molecules having a molecular weight of less than 5000, can bepresent). The term “pure” as used herein preferably has the samenumerical limits as “purified” immediately above.

A therapeutically “effective” amount means an amount of agent effectiveto prevent, alleviate or ameliorate symptoms of disease or prolong thesurvival of the subject being treated. Therapeutically effective dosagesmay be determined by using in vitro and in vivo methods, such as thosedescribed herein.

A “recombinant nucleic acid” is any nucleic acid that has been placedadjacent to another nucleic acid by recombinant DNA techniques. A“recombinant nucleic acid” also includes any nucleic acid that has beenplaced next to a second nucleic acid by a laboratory genetic techniquesuch as, for example, transformation and integration, transposon hoppingor viral insertion. In general, a recombined nucleic acid is notnaturally located adjacent to the second nucleic acid.

The term “recombinant protein” refers to a protein that is produced byexpression from a recombinant nucleic acid.

A “subject” is any organism of interest, generally a mammalian subject,such as a mouse, and preferably a human subject.

As used herein, the term “NSAID” includes, but is not limited to, thoseagents which inhibit cyclooxygenase, the enzyme responsible for thebiosyntheses of the prostaglandins and certain autocoid inhibitors,including inhibitors of the various isoenzymes of cyclooxygenase(including, but not limited to, cyclooxygenase-1 and -2), such as thecommercially available NSAIDs aceclofenac, acemetacin, acetaminophen,acetaminosalol, acetyl-salicylic acid (aspirin),acetyl-salicylic-2-amino-4-picoline-acid, 5-aminoacetylsalicylic acid,alclofenac, aminoprofen, amfenac, ampyrone, ampiroxicam, anileridine,bendazac, benoxaprofen, bermoprofen, a-bisabolol, bromfenac,5-bromosalicylic acid acetate, bromosaligenin, bucloxic acid, butibufen,carprofen, celecoxib, chromoglycate, cinmetacin, clindanac, clopirac,sodium diclofenac, diflunisal, ditazol, droxicam, enfenamic acid,etodolac, etofenamate, felbinac, fenbufen, fenclozic acid, fendosal,fenoprofen, fentiazac, fepradinol, flufenac, flufenamic acid, flunixin,flunoxaprofen, flurbiprofen, glutametacin, glycol salicylate, ibufenac,ibuprofen, ibuproxam, indomethacin, indoprofen, isofezolac, isoxepac,isoxicam, ketoprofen, ketorolac, lomoxicam, loxoprofen, meclofenamicacid, mefenamic acid, meloxicam, mesalamine, metiazinic acid, mofezolac,montelukast, mycophenolic acid, nabumetone, naproxen, niflumic acid,nimesulide, olsalazine, oxaceprol, oxaprozin, oxyphenbutazone,paracetamol, parsalmide, perisoxal, phenyl-acethyl-salicylate,phenylbutazone, phenylsalicylate, pyrazolac, piroxicam, pirprofen,pranoprofen, protizinic acid, resveratrol, salacetamide, salicylamide,salicylamide-O-acetyl acid, salicylsulphuric acid, salicin,salicylamide, salsalate, sulindac, suprofen, suxibutazone, tamoxifen,tenoxicam, theophylline, tiaprofenic acid, tiaramide, ticlopridine,tinoridine, tolfenamic acid, tolmetin, tropesin, xenbucin, ximoprofen,zaltoprofen, zomepirac, tomoxiprol, zafirlukast, rofecoxib andcyclosporine. Additionally, The Merck Manual, 16th Edition, MerckResearch Laboratories (1990) pp 1308-1309, as well as ThePharmacological Basis of Therapeutics, 9th edition, Macmillan PublishingCo., 1996, pp 617-655, provide well known examples of NSAIDs.

As used herein, conditions that are responsive to NSAID treatment (also“NSAID-responsive conditions”) refers to any disease, disorder orcondition that may be treated in a subject in need thereof with NSAIDadministration. NSAID-responsive conditions also include colon neoplasiaincluding, but not limited to, colon adenoma and colon cancer therapyand prevention, as described herein. Additional examples ofNSAID-responsive diseases and disorders include, but are not limited to,pain and inflammation.

In some embodiments, an “NSAID-responsive condition” also refers to acondition that is NSAID-resistant, but that becomes NSAID-responsive asa result of the methods disclosed herein. For example, the presentdisclosure is useful for treating an “NSAID-responsive condition”according to the methods described herein, even though the condition isin fact NSAID-resistant. That is, the methods disclosed herein turns anNSAID-resistant condition into an NSAID-responsive condition.Accordingly, it should be understood that “NSAID-responsive” refers toan NSAID-resistant condition that can be made NSAID-responsive.

Additionally, the present method can be used to treat anNSAID-responsive condition or subject in which the subject is alreadyNSAID-responsive. For example, the subject may be relativelyunresponsive to NSAID therapy, but not completely resistant. In otherembodiments, the present method can be used to treat a subject who isalready NSAID-responsive, but may benefit from the present invention.

As described herein, “NSAID-resistant” refers to a subject whose 15-PDGHlevel or activity is a fraction (or percentage) of the average 15-PDGHlevel or activity measured in a healthy population. The level oractivity of 15-PGDH from an NSAID-resistant subject sample is less than90%, 80%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, or 5% of the level or activity of 15-PGDH determined from a healthypopulation. As described herein, the level of 15-PGDH is a value of anamount of 15-PGDH protein or nucleic acid measured as a quantified unit.In certain embodiments, the activity of 15-PGDH is a measured unit ofenzymatic activity of 15-PGDH on a known substrate, e.g., PGE₂. In otherembodiments, such levels are average values measured from a pool of ahealthy population.

As used herein, the term “pain” includes all types of pain. Painincludes, but is not limited to, chronic pain, such as arthritis pain(e.g., pain associated with osteoarthritis and rheumatoid arthritis),neuropathic pain, and post-operative pain, chronic lower back pain,cluster headaches, herpes neuralgia, phantom limb pain, central pain,dental pain, neuropathic pain, opioid-resistant pain, visceral pain,surgical pain, bone injury pain, pain during labor and delivery, painresulting from burns, including sunburn, post partum pain, migraine,angina pain, and genitourinary tract-related pain including cystitis.The term also refers to nociceptive pain or nociception. The term alsorefers to persistent pain, such as, but not limited to, neuropathicpain, diabetic neuropathy, fibromyalgia, pain associated with somatoformdisorders, arthritic pain, cancer pain, neck pain, shoulder pain, backpain, cluster headaches, tension-type headache, migraine, herpesneuralgia, phantom limb pain, central pain, dental pain, paintraditionally resistant to treatment with NSAIDs, and post-operativepain. The term pain also refers to the pain associated with theinflammation-related diseases and disorders described herein.

The term “inflammation” and “inflammation-related disease and disorder”will be understood by those skilled in the art to include any conditioncharacterized by a localized protective response elicited by injury ordestruction of tissues resulting from any of the causes mentionedhereinbefore, and which is manifest by heat, swelling, pain, redness,dilation of blood vessels and/or increased blood flow, invasion of theaffected area by white blood cells, loss of function and/or any othersymptoms known to be associated with the inflammatory condition. Theterm will thus be understood to include, inter alia, acute, chronic,ulcerative, specific, allergic and necrotic inflammation, as well as allother forms of inflammation known to those skilled in the art. The termalso includes arthritis (including osteoarthritis and rheumatoidarthritis), inflammatory bowel disease, eczema, psoriasis, atopicdermatitis, psoriatic arthropathy and asthma, post operativeinflammation, dental inflammation, acute and chronic ocular inflammatorydiseases, conjunctivitis. The compounds and compositions of the subjectinvention advantageously can block the immunogenic inflammatory pathway,thereby providing a method for inhibiting immunogenic inflammation.Accordingly, the subject compounds and compositions can be useful in thetreatment of neurogenic inflammation, present in different processes,such as diabetes, asthma, cystitis, gingivitis, migraine, dermatitis,rhinitis, psoriasis, inflammation of sciatic and lumbar nerves,gastrointestinal processes, ocular inflammation, and acute allergicresponse, asthma, rheumatoid arthritis, osteoarthritis and otherinflammatory conditions involving acute and/or chronic jointinflammation in a subject, preferably mammalian, more preferably human.

Overview

It has been shown that 15-prostaglandin dehydrogenase (also known as15-hydroxyprostaglandin dehydrogenase; 15-PGDH), a prostaglandindegrading enzyme, functions as an endogenous inhibitor of the colonicCOX-2 pathway and as a colon tumor suppressor gene (Yan, M. et al.,(2004) Proc Natl Acad Sci USA 101, 17468-73; Myung, S. J. et al., (2006)Proc Natl Acad Sci U S A 103, 12098-102). 15-PGDH is highly expressed innormal colon mucosa, but expression is ubiquitously lost in human coloncancers (Yan, M. et al., (2004) Proc Natl Acad Sci U S A 101, 17468-73;Backlund, M. G. et al., (2005) J Biol Chem 280, 3217-23). Knocking outthe murine 15-PGDH gene markedly sensitizes colon tumor induction by thecarcinogen azoxymethane (AOM) in normally resistant C57BL/6J mice(Myung, S. J. et al., (2006) Proc Natl Acad Sci U S A 103, 12098-102).

The disclosure provided herein shows, in part, that the adenomaprevention activity of the COX-2 inhibitor celecoxib requires theconcomitant presence of the 15-prostaglandin dehydrogenase (15-PGDH)tumor suppressor gene, and that loss of 15-PGDH expression impartsresistance to celecoxib's anti-tumor effects. For example, the adenomapreventive activity of celecoxib is abrogated in mice geneticallylacking 15-PGDH. In FVB mice, celecoxib prevents 85% of azoxymethane(AOM) induced tumors >1 mm in size, but is essentially inactive inpreventing tumor induction in 15-PGDH null animals. Indeed, celecoxibtreated 15-PGDH null animals develop more tumors than do celecoxib naïvewild-type mice. In parallel with the loss of tumor prevention activity,celecoxib-mediated suppression of colonic PGE2 levels is also markedlyattenuated in 15-PGDH null versus wild-type mice. Additionally, aspredicted by the murine models, humans with low colonic 15-PGDH levelsalso demonstrate celecoxib resistance.

Specifically, in a colon adenoma prevention trial, in all cases tested,individuals who developed new adenomas while on celecoxib treatment werealso found as having low colonic 15-PGDH. Finally, because drugs thatinhibit COX activity are among the most common medications prescribedfor relief of pain and inflammation, it is contemplated that low 15-PGDHlevels also impart clinical resistance to these therapeutic activitiesand, therefore, the therapeutic benefits of increasing 15-PGDH can beextended beyond cancer therapy.

The interactions between pharmacologic regulation of colonicprostaglandins by celecoxib and the genetic regulation of colonicprostaglandins by 15-PGDH are disclosed herein. Pharmacologic inhibitorsof the prostaglandin-synthesizing COX-2 oncogene prevent the developmentof premalignant human colon adenomas. Resistance to treatment, however,is common. Accordingly, the invention relates, in part, to a method ofidentifying subjects who would be responsive to non-steroidalinflammatory drug (NSAID) therapy, comprising: (i) obtaining abiological sample from said subject; and (ii) measuring 15-PGDH levelsor 15-PGDH activity, wherein normal or increased level or activity of15-PGDH indicates that the subject would be responsive to NSAID therapy.The subject may be a subject at risk of developing colon neoplasia(e.g., based on family history), or a subject at risk of colon adenomarelapse. Further, the subject may be any patient who is undergoing orabout to undergo NSAID therapy for any NSAID-responsive condition. Thatis, the subject may be one who is about to undergo NSAID therapy, andthe method disclosed herein may be used to determine whether the subjectwill benefit from the NSAID therapy (i.e., determines if the subjectmight be resistant to NSAID therapy).

Another aspect of the invention relates to a method of determiningwhether a subject is predisposed to developing NSAID resistance in atherapy, comprising: (i) obtaining a biological sample from saidsubject; and (ii) measuring 15-PGDH levels or 15-PGDH activity, whereina reduced level or activity of 15-PGDH in the subject relative a normalsample indicates that the subject is predisposed to developing NSAIDresistance in the therapy. The normal sample may be derived from ahealthy subject. Alternatively, the reference sample may be derived froma subject who has previously suffered from colon neoplasia, but hasundergone NSAID therapy with no colon adenoma relapse (i.e., a subjectresponsive to NSAID therapy). For any of these reference values, one ofordinary skill in the art would understand that the value may be a meanor median of a representative pool of subjects.

Another aspect of the invention relates to a method of treating anyNSAID-responsive condition comprising administering an effective amountof an NSAID and an agent that upregulates 15-PGDH. In particular, themethod of treating an NSAID-responsive condition applies to a subjectwho is NSAID-resistant or a subject who, through the methods describedherein, was determined to be resistant to NSAID therapy.

Another aspect of the invention relates to a method of decreasing NSAIDresistance in a patient, comprising administering: (i) an effectiveamount of a compound that increases 15-PGDH levels or 15-PGDH activity;or (ii) an effective amount of 15-PGDH protein, cDNA, or an activefragment thereof. The patient may be a subject at risk of developingcolon neoplasia (e.g., based on family history), or a subject at risk ofcolon adenoma relapse, but is suspected of being resistant to NSAIDtherapy. Further, the patient may be any subject who is undergoing orabout to undergo NSAID therapy for any NSAID-responsive condition, butwho experiences NSAID resistance.

In one aspect, the present invention provides a method of identifying asubject who is at risk for developing colon neoplasia, comprising: (i)obtaining a non-neoplastic sample from said subject; and (ii) measuring15-PGDH levels or 15-PGDH activity, wherein a level or activity of15-PGDH that is less than 50% of the level or activity of 15-PGDH from ahealthy population indicates that the subject is at risk for developingcolon neoplasia. The normal sample as disclosed herein may be derivedfrom a healthy subject. Alternatively, the normal sample may be derivedfrom a subject who has previously suffered from colon neoplasia, but hasundergone NSAID therapy with no colon adenoma relapse (i.e., a subjectresponsive to NSAID therapy). For any of these normal reference values,one of ordinary skill in the art would understand that the value may bea mean or median of a representative pool of subjects.

One of ordinary skill in the art will recognize that the foregoingaspects and embodiments may be combined.

Measurement/Detection of 15-PGDH

In certain embodiments, the invention provides methods for detectingmolecular markers, such as proteins or nucleic acid transcripts of15-PGDH described herein. In certain embodiments, a method of theinvention comprises providing a biological sample and probing thebiological sample for the presence of a 15-PGDH. Information regardingthe presence or absence of 15-PGDH, and optionally the quantitativelevel of 15-PGDH, may then be used to draw inferences about the natureof the biological sample and, if the biological sample was obtained froma subject, the health state of the subject.

Samples for use with the methods described herein may be essentially anybiological material of interest. For example, a sample may be a tissuesample from a subject, a fluid sample from a subject, a solid orsemi-solid sample from a subject, a primary cell culture or tissueculture of materials derived from a subject, cells from a cell line, ormedium or other extracellular material from a cell or tissue culture, ora xenograft (meaning a sample of a colon cancer from a first subject,e.g. a human, that has been cultured in a second subject, e.g. animmunocompromised mouse). In certain embodiments, the sample iscolon/rectal tissue, e.g., ascending colon, transverse colon, descendingcolon, sigmoid colon, or redundant colon.

The term “sample” as used herein is intended to encompass both abiological material obtained directly from a subject (which may bedescribed as the primary sample) as well as any manipulated forms orportions of a primary sample. For example, in certain embodiments, afluid sample may be a blood sample. In this case, the term sample isintended to encompass not only the blood as obtained directly from thepatient but also fractions of the blood, such as plasma, serum, cellfractions (e.g. platelets, erythrocytes, lymphocytes), proteinpreparations, nucleic acid preparations, etc. A sample may also beobtained by contacting a biological material with an exogenous liquid,resulting in the production of a lavage liquid containing some portionof the contacted biological material. Furthermore, the term “sample” isintended to encompass the primary sample after it has been mixed withone or more additive, such as preservatives, chelators, anti-clottingfactors, etc. In certain embodiments, a fluid sample may be a urinesample. In certain embodiments, a preferred solid or semi-solid samplemay be a stool sample. In certain embodiments, a preferred tissue samplemay be a biopsy from a tissue known to harbor or suspected of harboringa colon neoplasia. In certain embodiments, a preferred cell culturesample may be a sample comprising cultured cells of a colon cancer cellline, such as a cell line cultured from a metastatic colon cancer tumoror a colon-derived cell line lacking a functional TGF-β, TGF-β receptoror TGF-β signaling pathway. A subject is preferably a human subject, butit is expected that the molecular markers disclosed herein, andparticularly their homologs from other animals, are of similar utilityin other animals. In certain embodiments, it may be possible to detect amarker directly in an organism without obtaining a separate portion ofbiological material. In such instances, the term sample is intended toencompass that portion of biological material that is contacted with areagent or device involved in the detection process.

In certain aspects, samples of the subject methods can be collected frompatients. These patients may be subjects who have been determined tohave a high risk of having a condition of interest based on theirpersonal or family history. Other patients include subjects who areknown to have an NSAID-responsive condition and for whom the test isbeing used to determine the effectiveness of therapy or treatment theyare receiving. Also, samples could be collected from healthy subjectswho are having a test as part of a routine examination, or to establishbaseline levels (e.g., a control or reference level) of the biomarker.Alternatively, samples could be collected from subjects who are known tobe responsive to NSAID therapy.

Samples should generally be prepared in a manner that is consistent withthe detection system to be employed. For example, a sample to be used ina protein detection system should generally be prepared in the absenceof proteases. Likewise, a sample to be used in a nucleic acid detectionsystem should generally be prepared in the absence of nucleases. In manyinstances, a sample for use in an antibody-based detection system willnot be subjected to substantial preparatory steps. For example, urinemay be used directly, as may saliva and blood, although blood will, incertain preferred embodiments, be separated into fractions such asplasma and serum.

Assays can be used to determine presence or absence of a marker (e.g.,mRNA or protein) in a sample as well as the quantity of a marker in asample. The amount of the marker can be determined by comparing to astandard. A standard can be, e.g., an amount or level known to bepresent or predetermined in a control sample (e.g., a healthy subject, asubject already responsive to NSAID therapy who does not require thedisclosed methods, or a subject who has successfully undergone an NSAIDtherapy, i.e., a subject responsive to NSAID therapy). It is understoodthat the test amount of the marker need not be measured in absoluteunits, as long as the unit of measurement can be compared relative to acontrol.

Measurement/Detection of Polynucleotides of 15-PGDH

Those of skill in the art will recognize that the measurement ordetection of the expression of 15-PGDH polynucleotides has many uses.For example, as discussed herein, measuring the 15-PGDH levels in apatient is useful for diagnosing colon cancer, or determining whether asubject is predisposed to developing colon cancer. Additionally,measuring the 15-PGDH levels in a subject is useful for determiningwhether the subject may benefit from the colon cancer preventiveproperties of NSAIDs. The subject in question may already be afflictedwith colon cancer, or the subject may be at risk of developing coloncancer. In some embodiments, the subject was previously afflicted withcolon cancer and is in a state of remission. Moreover, as describedherein, detection of gene expression is useful to identify modulators of15-PGDH expression.

In certain embodiments, a method of the invention comprises detectingthe presence of a 15-PGDH nucleic acid, such as an mRNA, in a sample.Optionally, the method involves obtaining a quantitative measure of theexpressed nucleic acid in the sample. In view of this specification, oneof skill in the art will recognize a wide range of techniques that maybe employed to detect and optionally quantify the presence of a nucleicacid. Nucleic acid detection systems generally involve preparing apurified nucleic acid fraction of a sample, and subjecting the sample toa direct detection assay or an amplification process followed by adetection assay. Amplification may be achieved, for example, bypolymerase chain reaction (PCR), reverse transcriptase (RT) and coupledRT-PCR. Detection of a nucleic acid is generally accomplished by probingthe purified nucleic acid fraction with a probe that hybridizes to thenucleic acid of interest, and in many instances detection involves anamplification as well. Northern blots, dot blots, microarrays,quantitative PCR and quantitative RT-PCR are all well known methods fordetecting a nucleic acid in a sample.

In certain embodiments, the invention provides nucleic acid probes thatbind specifically to a 15-PGDH nucleic acid. Such probes may be labeledwith, for example, a fluorescent moiety, a radionuclide, an enzyme or anaffinity tag such as a biotin moiety. For example, the TaqMan® systememploys nucleic acid probes that are labeled in such a way that thefluorescent signal is quenched when the probe is free in solution andbright when the probe is incorporated into a larger nucleic acid.

A variety of methods of specific DNA and RNA measurement that usenucleic acid hybridization techniques are known to those of skill in theart (see, Sambrook, supra). Some methods involve an electrophoreticseparation (e.g., Southern blot for detecting DNA, and Northern blot fordetecting RNA), but measurement of DNA and RNA can also be carried outin the absence of electrophoretic separation (e.g., by dot blot).Southern blot of genomic DNA (e.g., from a human) can be used forscreening for restriction fragment length polymorphism (RFLP) to detectthe presence of a genetic disorder affecting a 15-PGDH polypeptide ofthe invention.

The selection of a nucleic acid hybridization format is not critical. Avariety of nucleic acid hybridization formats are known to those skilledin the art. For example, common formats include sandwich assays andcompetition or displacement assays. Hybridization techniques aregenerally described in Hames and Higgins Nucleic Acid Hybridization, APractical Approach, IRL Press (1985); Gall and Pardue, Proc. Natl. Acad.Sci. U.S.A., 63:378-383 (1969); and John et al. Nature, 223:582-587(1969).

Detection of a hybridization complex may require the binding of asignal-generating complex to a duplex of target and probepolynucleotides or nucleic acids. Typically, such binding occurs throughligand and anti-ligand interactions as between a ligand-conjugated probeand an anti-ligand conjugated with a signal. The binding of the signalgeneration complex is also readily amenable to accelerations by exposureto ultrasonic energy.

The label may also allow indirect detection of the hybridizationcomplex. For example, where the label is a hapten or antigen, the samplecan be detected by using antibodies. In these systems, a signal isgenerated by attaching fluorescent or enzyme molecules to the antibodiesor in some cases, by attachment to a radioactive label (see, e.g.,Tijssen, “Practice and Theory of Enzyme Immuoassays,” LaboratoryTechniques in Biochemistry and Molecular Biology, Burdon and vanKnippenberg Eds., Elsevier (1985), pp. 9-20).

The probes are typically labeled either directly, as with isotopes,chromophores, lumiphores, chromogens, or indirectly, such as withbiotin, to which a streptavidin complex may later bind. Thus, thedetectable labels used in the assays of the present invention can beprimary labels (where the label comprises an element that is detecteddirectly or that produces a directly detectable element) or secondarylabels (where the detected label binds to a primary label, e.g., as iscommon in immunological labeling). Typically, labeled signal nucleicacids are used to detect hybridization. Complementary nucleic acids orsignal nucleic acids may be labeled by any one of several methodstypically used to detect the presence of hybridized polynucleotides. Themost common method of detection is the use of autoradiography with ³H,¹²⁵I, ³⁵S, ¹⁴C or ³²P-labeled probes or the like.

Other labels include, e.g., ligands that bind to labeled antibodies,fluorophores, chemiluminescent agents, enzymes, and antibodies that canserve as specific binding pair members for a labeled ligand. Anintroduction to labels, labeling procedures and detection of labels isfound in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nded., Springer Verlag, NY (1997); and in Haugland, Handbook ofFluorescent Probes and Research Chemicals, a combined handbook andcatalogue Published by Molecular Probes, Inc. (1996).

In general, a detector that monitors a particular probe or probecombination is used to detect the detection reagent label. Typicaldetectors include spectrophotometers, phototubes and photodiodes,microscopes, scintillation counters, cameras, film and the like, as wellas combinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons of skillin the art. Commonly, an optical image of a substrate comprising boundlabeling moieties is digitized for subsequent computer analysis.

The amount of, for example, an RNA is measured by quantifying the amountof label fixed to the solid support by binding of the detection reagent.Typically, the presence of a modulator during incubation will increaseor decrease the amount of label fixed to the solid support relative to acontrol incubation that does not comprise the modulator, or as comparedto a baseline established for a particular reaction type. Means ofdetecting and quantitating labels are well known to those of skill inthe art.

In some embodiments, the target nucleic acid or the probe is immobilizedon a solid support. Solid supports suitable for use in the assays of theinvention are known to those of skill in the art. As used herein, asolid support is a matrix of material in a substantially fixedarrangement.

A variety of automated solid-phase assay techniques are alsoappropriate. For instance, very large scale immobilized polymer arrays(VLSIPS™), i.e. Gene Chips or microarrays, available from Affymetrix,Inc. in Santa Clara, Calif. can be used to detect changes in expressionlevels of a plurality of genes involved in the same regulatory pathwayssimultaneously. See, Tijssen, supra., Fodor et al. (1991) Science, 251:767-777; Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719, andKozal et al. (1996) Nature Medicine 2(7): 753-759. Similarly, spottedcDNA arrays (arrays of cDNA sequences bound to nylon, glass or anothersolid support) can also be used to monitor expression of a plurality ofgenes.

Typically, the array elements are organized in an ordered fashion sothat each element is present at a specified location on the substrate.Because the array elements are at specified locations on the substrate,the hybridization patterns and intensities (which together create aunique expression profile) can be interpreted in terms of expressionlevels of particular genes and can be correlated with a particulardisease or condition or treatment. See, e.g., Schena et al., Science270: 467-470 (1995) and Lockhart et al., Nature Biotech. 14: 1675-1680(1996). The various methods of evaluating hybridization detection andspecificity are described in US 2006160076 A1, incoporated herein in itsentirety.

Detection of nucleic acids can also be accomplished, for example, byusing a labeled detection moiety that binds specifically to duplexnucleic acids (e.g., an antibody that is specific for RNA-DNA duplexes).One example uses an antibody that recognizes DNA-RNA heteroduplexes inwhich the antibody is linked to an enzyme (typically by recombinant orcovalent chemical bonding). The antibody is detected when the enzymereacts with its substrate, producing a detectable product. Coutlee etal. (1989) Analytical Biochemistry 181:153-162; Bogulayski (1986) et al.J. Immunol. Methods 89:123-130; Prooijen-Knegt (1982) Exp. Cell Res.141:397-407; Rudkin (1976) Nature 265:472-473, Stollar (1970) PNAS65:993-1000; Ballard (1982) Mol. Immunol. 19:793-799; Pisetsky andCaster (1982) Mol. Immunol. 19:645-650; Viscidi et al. (1988) J. Clin.Microbial. 41:199-209; and Kiney et al. (1989) J. Clin. Microbiol.27:6-12 describe antibodies to RNA duplexes, including homo andheteroduplexes. Kits comprising antibodies specific for DNA:RNA hybridsare available, e.g., from Digene Diagnostics, Inc. (Beltsville, Md.).

In addition to available antibodies, one of skill in the art can easilymake antibodies specific for nucleic acid duplexes using existingtechniques, or modify those antibodies that are commercially or publiclyavailable. In addition to the art referenced above, general methods forproducing polyclonal and monoclonal antibodies are known to those ofskill in the art (see, e.g., Paul (ed) Fundamental Immunology, ThirdEdition Raven Press, Ltd., NY (1993); Coligan Current Protocols inImmunology Wiley/Greene, NY (1991); Harlow and Lane Antibodies: ALaboratory Manual Cold Spring Harbor Press, NY (1989); Stites et al.(eds.) Basic and Clinical Immunology (4th ed.) Lange MedicalPublications, Los Altos, Calif., and references cited therein; GodingMonoclonal Antibodies: Principles and Practice (2d ed.) Academic Press,New York, N.Y., (1986); and Kohler and Milstein Nature 256: 495-497(1975)). Other suitable techniques for antibody preparation includeselection of libraries of recombinant antibodies in phage or similarvectors (see, Huse et al. Science 246:1275-1281 (1989); and Ward et al.Nature 341:544-546 (1989)). Specific monoclonal and polyclonalantibodies and antisera will usually bind with a K_(D) of at least about0.1 μM, preferably at least about 0.01 μM or better, and most typicallyand preferably, 0.001 μM or better.

The nucleic acids used in this invention can be either positive ornegative probes. Positive probes bind to their targets and the presenceof duplex formation is evidence of the presence of the target. Negativeprobes fail to bind to the suspect target and the absence of duplexformation is evidence of the presence of the target. For example, theuse of a wild type specific nucleic acid probe or PCR primers may serveas a negative probe in an assay sample where only the nucleotidesequence of interest is present.

The sensitivity of the hybridization assays may be enhanced through useof a nucleic acid amplification system that multiplies the targetnucleic acid being detected. Examples of such systems include thepolymerase chain reaction (PCR) system and the ligase chain reaction(LCR) system. Other methods recently described in the art are thenucleic acid sequence based amplification (NASBA, Cangene, Mississauga,Ontario) and Q Beta Replicase systems. These systems can be used todirectly identify mutants where the PCR or LCR primers are designed tobe extended or ligated only when a selected sequence is present.Alternatively, the selected sequences can be generally amplified using,for example, nonspecific PCR primers and the amplified target regionlater probed for a specific sequence indicative of a mutation. It isunderstood that various detection probes, including Taqman and molecularbeacon probes can be used to monitor amplification reaction products,e.g., in real time.

An alternative means for determining the level of expression of thenucleic acids of the present invention is in situ hybridization. In situhybridization assays are well known and are generally described inAngerer et al., Methods Enzymol. 152:649-660 (1987). In an in situhybridization assay, cells, preferentially human cells from thecerebellum or the hippocampus, are fixed to a solid support, typically aglass slide. If DNA is to be probed, the cells are denatured with heator alkali. The cells are then contacted with a hybridization solution ata moderate temperature to permit annealing of specific probes that arelabeled. The probes are preferably labeled with radioisotopes orfluorescent reporters.

Single nucleotide polymorphism (SNP) analysis is also useful fordetecting differences between alleles of 15-PGDH genes. 15-PGDH linkedSNPs are useful, for instance, for diagnosis of 15-PGDH-linked diseases(e.g., colon cancer) in a patient. For example, if an individual carriesat least one allele of a 15-PGDH -linked SNP, the individual is likelypredisposed for one or more of those diseases. If the individual ishomozygous for a disease-linked 15-PGDH SNP, the individual isparticularly predisposed for 15-PGDH-linked disease. In someembodiments, the SNP associated with the 15-PGDH-linked disease islocated within 300,000; 200,000; 100,000; 75,000; 50,000; or 10,000 basepairs of a polynucleotide encoding 15-PGDH.

Various real-time PCR methods including, e.g., Taqman or molecularbeacon-based assays (e.g., U.S. Pat. Nos. 5,210,015; 5,487,972; Tyagi etal., (1996) Nature Biotechnology 14:303; and PCT WO 95/13399) are usefulto monitor for the presence of absence of a SNP. Additional SNPdetection methods include, e.g., DNA sequencing, sequencing byhybridization, dot blotting, oligonucleotide array (DNA Chip)hybridization analysis, or are described in, e.g., U.S. Pat. No.6,177,249; Landegren et al., (1998) Genome Research, 8:769-776; Botsteinet al., (1980) Am J Human Genetics 32:314-331; Meyers et al., (1987)Methods in Enzymology 155:501-527; Keen et al., (1991) Trends inGenetics 7:5; Myers et al., (1985) Science 230:1242-1246; and Kwok etal., (1994) Genomics 23:138-144.

Immunological Measurement/Detection of 15-PGDH

In certain embodiments, a method of the invention comprises detectingthe levels of the 15-PGDH protein in a sample. Additionally, a method ofthe invention comprises detecting the activity level of 15-PGDH (e.g.,an assay which measures the enzymatic activity of 15-PGDH, as known inthe art). Optionally, the method involves obtaining a quantitativemeasure of the 15-PGDH protein in the sample. In view of thisspecification, one of skill in the art will recognize a wide range oftechniques that may be employed to detect and optionally quantify thepresence of a protein. A general overview of the applicable technologycan be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988).In some embodiments, the 15-PGDH protein is detected with an antibody.In many embodiments, an antibody-based detection assay involves bringingthe sample and the antibody into contact so that the antibody has anopportunity to bind to proteins having the corresponding epitope. Inmany embodiments, an antibody-based detection assay also typicallyinvolves a system for detecting the presence of antibody-epitopecomplexes, thereby achieving a detection of the presence of the proteinshaving the corresponding epitope. Antibodies may be used in a variety ofdetection techniques, including enzyme-linked immunosorbent assays(ELISAs), immunoprecipitations, Western blots.

Antibody-independent techniques for identifying a protein may also beemployed. For example, mass spectroscopy, particularly coupled withliquid chromatography, permits detection and quantification of largenumbers of proteins in a sample. Two-dimensional gel electrophoresis mayalso be used to identify proteins, and may be coupled with massspectroscopy or other detection techniques, such as N-terminal proteinsequencing. RNA aptamers with specific binding for the protein ofinterest may also be generated and used as a detection reagent.

Methods for producing polyclonal and monoclonal antibodies that reactspecifically with a protein of interest or other immunogen are known tothose of skill in the art (see, e.g., Coligan, supra; and Harlow andLane, supra; Stites et al., supra and references cited therein; Goding,supra; and Kohler and Milstein Nature, 256:495-497 (1975)). Suchtechniques include antibody preparation by selection of antibodies fromlibraries of recombinant antibodies in phage or similar vectors (see,Huse et al., supra; and Ward et al., supra). For example, in order toproduce antisera for use in an immunoassay, the protein of interest oran antigenic fragment thereof, is isolated as described herein. Forexample, a recombinant protein is produced in a transformed cell line.An inbred strain of mice or rabbits is immunized with the protein usinga standard adjuvant, such as Freund's adjuvant, and a standardimmunization protocol. Alternatively, a synthetic peptide derived fromthe 15-PGDH sequence is conjugated to a carrier protein and used as animmunogen.

Polyclonal sera are collected and titered against the immunogen in animmunoassay, for example, a solid phase immunoassay with the immunogenimmobilized on a solid support. Polyclonal antisera with a titer of 10⁴or greater are selected and tested for their crossreactivity againstnon-15-PGDH proteins or even other homologous proteins from otherorganisms, using a competitive binding immunoassay. Specific monoclonaland polyclonal antibodies and antisera will usually bind with a K_(D) ofat least about 0.1 mM, more usually at least about 1 μM, preferably atleast about 0.1 μM or better, and most preferably, 0.01 μM or better.

A number of proteins of the invention comprising immunogens may be usedto produce antibodies specifically or selectively reactive with theproteins of interest. Recombinant protein is the preferred immunogen forthe production of monoclonal or polyclonal antibodies. Naturallyoccurring protein may also be used either in pure or impure form.Synthetic peptides made using the protein sequences described herein mayalso be used as an immunogen for the production of antibodies to theprotein.

Recombinant protein can be expressed in eukaryotic or prokaryotic cellsand purified as generally described supra. The product is then injectedinto an animal capable of producing antibodies. Either monoclonal orpolyclonal antibodies may be generated for subsequent use inimmunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen, preferably a purified protein,is mixed with an adjuvant and animals are immunized. The animal's immuneresponse to the immunogen preparation is monitored by taking test bleedsand determining the titer of reactivity to 15-PGDH. When appropriatelyhigh titers of antibody to the immunogen are obtained, blood iscollected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theprotein can be done if desired (see, Harlow and Lane, supra).

Monoclonal antibodies may be obtained using various techniques familiarto those of skill in the art. Typically, spleen cells from an animalimmunized with a desired antigen are immortalized, commonly by fusionwith a myeloma cell (see, Kohler and Milstein, Eur. J. Immunol.6:511-519 (1976)). Alternative methods of immortalization include, e.g.,transformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. Alternatively, one may isolate DNA sequences thatencode a monoclonal antibody or a binding fragment thereof by screeninga DNA library from human B cells according to the general protocoloutlined by Huse et al., supra.

Once target immunogen-specific antibodies are available, the immunogencan be measured by a variety of immunoassay methods with qualitative andquantitative results available to the clinician. For a review ofimmunological and immunoassay procedures in general see, Stites, supra.Moreover, the immunoassays of the present invention can be performed inany of several configurations, which are reviewed extensively in MaggioEnzyme Immunoassay, CRC Press, Boca Raton, Fla. (1980); Tijssen, supra;and Harlow and Lane, supra.

Immunoassays to measure target proteins in a human sample may use apolyclonal antiserum that was raised to the protein (e.g., 15-PGDH) or afragment thereof. This antiserum is selected to have lowcross-reactivity against non-15-PGDH proteins and any suchcross-reactivity is removed by immunoabsorption prior to use in theimmunoassay.

In some embodiments, a protein of interest is detected and/or quantifiedusing any of a number of well-known immunological binding assays (see,e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).For a review of the general immunoassays, see also Asai Methods in CellBiology Volume 37: Antibodies in Cell Biology, Academic Press, Inc. NY(1993); Stites, supra. Immunological binding assays (or immunoassays)typically utilize a “capture agent” to specifically bind to and oftenimmobilize the analyte (in this case 15-PGDH of the present invention,or antigenic subsequences thereof). The capture agent is a moiety thatspecifically binds to the analyte. In a preferred embodiment, thecapture agent is an antibody that specifically binds, for example, a15-PGDH polypeptide of the invention. The antibody (e.g., anti-15-PGDHantibody) may be produced by any of a number of means well known tothose of skill in the art and as described above.

Immunoassays also often utilize a labeling agent to bind specifically toand label the binding complex formed by the capture agent and theanalyte. The labeling agent may itself be one of the moieties comprisingthe antibody/analyte complex. Alternatively, the labeling agent may be athird moiety, such as another antibody, that specifically binds to theantibody/protein complex.

In a preferred embodiment, the labeling agent is a second antibodybearing a label. Alternatively, the second antibody may lack a label,but it may, in turn, be bound by a labeled third antibody specific toantibodies of the species from which the second antibody is derived. Thesecond antibody can be modified with a detectable moiety, such asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constantregions, such as protein A or protein G, can also be used as the labelagents. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (see,generally, Kronval, et al. J Immunol., 111: 1401-1406 (1973); andAkerstrom, et al. J Immunol., 135:2589-2542 (1985)).

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. The incubation time will depend upon the assay format, analyte,volume of solution, concentrations, and the like. Usually, the assayswill be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 10° C. to 40° C.

Immunoassays for detecting proteins or analytes of interest from tissuesamples may be either competitive or noncompetitive. Noncompetitiveimmunoassays are assays in which the amount of captured protein oranalyte is directly measured. In one preferred “sandwich” assay, forexample, the capture agent (e.g. 15-PGDH antibodies) can be bounddirectly to a solid substrate where it is immobilized. These immobilizedantibodies then capture the 15-PGDH present in the test sample. The15-PGDH thus immobilized is then bound by a labeling agent, such as asecond anti-15-PGDH antibody bearing a label. Alternatively, the secondantibody may lack a label, but it may, in turn, be bound by a labeledthird antibody specific to antibodies of the species from which thesecond antibody is derived. The second can be modified with a detectablemoiety, such as biotin, to which a third labeled molecule canspecifically bind, such as enzyme-labeled streptavidin.

In competitive assays, the amount of protein or analyte present in thesample is measured indirectly by measuring the amount of an added(exogenous) protein or analyte (e.g., the 15-PGDH of interest) displaced(or competed away) from a specific capture agent (e.g., antibodiesraised to 15-PGDH) by the protein or analyte present in the sample. Theamount of immunogen bound to the antibody is inversely proportional tothe concentration of immunogen present in the sample. In a particularlypreferred embodiment, the antibody is immobilized on a solid substrate.The amount of analyte may be detected by providing a labeled analytemolecule. It is understood that labels can include, e.g., radioactivelabels as well as peptide or other tags that can be recognized bydetection reagents such as antibodies.

Immunoassays in the competitive binding format can be used forcross-reactivity determinations. For example, the protein encoded by thesequences described herein can be immobilized on a solid support.Proteins are added to the assay and compete with the binding of theantisera to the immobilized antigen. The ability of the above proteinsto compete with the binding of the antisera to the immobilized proteinis compared to that of the protein encoded by any of the sequencesdescribed herein. The percent cross-reactivity for the above proteins iscalculated, using standard calculations. Those antisera with less than10% cross-reactivity with each of the proteins listed above are selectedand pooled. The cross-reacting antibodies are optionally removed fromthe pooled antisera by immunoabsorption with the considered proteins,e.g., distantly related homologs.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps a protein of the present invention, to theimmunogen protein. In order to make this comparison, the two proteinsare each assayed at a wide range of concentrations and the amount ofeach protein required to inhibit 50% of the binding of the antisera tothe immobilized protein is determined. If the amount of the secondprotein required is less than 10 times the amount of the proteinpartially encoded by a sequence herein that is required, then the secondprotein is said to specifically bind to an antibody generated to animmunogen consisting of the target protein.

In other embodiments, western blot (immunoblot) analysis is used todetect and quantify the presence of a 15-PGDH in the sample. Thetechnique generally comprises separating sample proteins by gelelectrophoresis on the basis of molecular weight, transferring theseparated proteins to a suitable solid support (e.g., a nitrocellulosefilter, a nylon filter, or a derivatized nylon filter) and incubatingthe sample with the antibodies that specifically bind the protein ofinterest. For example, the anti-15-PGDH antibodies specifically bind tothe 15-PGDH on the solid support. These antibodies may be directlylabeled or alternatively may be subsequently detected using labeledantibodies (e.g., labeled sheep anti-mouse antibodies) that specificallybind to the antibodies against the protein of interest.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see, Monroe et al.(1986) Amer. Clin. Prod. Rev. 5:3441).

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most labels useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., Dynabeads™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), and calorimetric labels such ascolloidal gold or colored glass or plastic (e.g. polystyrene,polypropylene, latex, etc.) beads.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on the sensitivity required, the ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Themolecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorescent compound.A variety of enzymes and fluorescent compounds can be used with themethods of the present invention and are well-known to those of skill inthe art (for a review of various labeling or signal producing systemswhich may be used, see, e.g., U.S. Pat. No. 4,391,904).

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple colorimetriclabels may be detected directly by observing the color associated withthe label. Thus, in various dipstick assays, conjugated gold oftenappears pink, while various conjugated beads appear the color of thebead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need to be labeled and the presence ofthe target antibody is detected by simple visual inspection.

Identification of Modulators of 15-PGDH

Modulators of 15-PGDH, i.e. agonists or antagonists of 15-PGDH activity,or 15-PGDH polypeptide or polynucleotide expression, are useful fortreating a number of human diseases, including cancer. For example,administration of 15-PGDH upregulators can be used to treat patientswith colon neoplasia, e.g., colon cancer or colon adenoma, or patientswith a history of colon neoplasia with resistance to NSAID therapy forneoplasia therapy or neoplasia preventive therapy. Further,administration of 15-PGDH upregulators can be used to treat subjectshaving an NSAID-responsive condition, as described herein. In certainembodiments, 15-PGDH upregulators enhance NSAID-responsiveness insubjects who are relatively unresponsive to NSAID treatment.

“Inhibitors,” “activators,” and “modulators” of 15-PGDH expression or of15-PGDH activity are used to refer to inhibitory, activating, ormodulating molecules, respectively, identified using in vitro and invivo assays for 15-PGDH expression or 15-PGDH activity, e.g., ligands,agonists, antagonists, and their homologs and mimetics. The term“modulator” includes inhibitors and activators. Inhibitors are agentsthat, e.g., inhibit expression of 15-PGDH or bind to, partially ortotally block stimulation, decrease, prevent, delay activation,inactivate, desensitize, or down regulate the activity of 15-PGDH, e.g.,antagonists. Activators are agents that, e.g., induce or activate theexpression of a 15-PGDH or bind to, stimulate, increase, open, activate,facilitate, or enhance activation, sensitize or up regulate the activityof 15-PGDH, e.g., agonists. Modulators include naturally occurring andsynthetic ligands, small chemical molecules, and the like. Accordingly,a 15-PGDH activator may include an agent that enhances the activity orexpression of 15-PGDH directly. In other embodiments, a 15-PGDHactivator may include an agent that enhances the expression of 15-PGDHindirectly. For example, as described herein, turning off thebeta-catenin transcription factor markedly induces the expression ofboth 15-PGDH mRNA and protein (FIG. 4).

Described herein are assays for identifying inhibitors and activatorsSuch assays include, e.g., applying putative modulator compounds tocells expressing 15-PGDH and then determining the functional effects on15-PGDH activity. Samples or assays comprising 15-PGDH that are treatedwith a potential activator, inhibitor, or modulator are compared tocontrol samples without the inhibitor, activator, or modulator toexamine the extent of effect. Control samples (untreated withmodulators) are assigned a relative 15-PGDH activity value of 100%.Inhibition of 15-PGDH is achieved when the 15-PGDH activity valuerelative to the control is about 80%, optionally 50% or 25, 10%, 5% or1%. Activation of 15-PGDH is achieved when the 15-PGDH activity orexpression value relative to the control is 105%, optionally 110%,optionally 125%, optionally 150%, optionally 200, 300%, 400%, 500%, or1000-3000% or more higher.

Agents that Modulate 15-PGDH

The agents tested as modulators of 15-PGDH can be any small chemicalcompound, or a biological entity, such as a protein, sugar, nucleic acidor lipid. Typically, test compounds will be small chemical molecules andpeptides. Essentially any chemical compound can be used as a potentialmodulator or ligand in the assays of the invention. The assays aredesigned to screen large chemical libraries by automating the assaysteps and providing compounds from any convenient source to assays,which are typically run in parallel (e.g., in microtiter formats onmicrotiter plates in robotic assays). Modulators also include agentsdesigned to increase the level of 15-PGDH mRNA or the level oftranslation from an mRNA. It will be appreciated that there are manysuppliers of chemical compounds, including Sigma (St. Louis, Mo.),Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), FlukaChemika-Biochemica Analytika (Buchs, Switzerland) and the like. Someexamples of small molecules that induce 15-PGDH mRNA levels include, butare not limited to, erlotinib (Backlund, M. G. et al., (2005) J BiolChem 280, 3217-23), butyrate (Backlund, M. G., et al, (2008) Cancer Res68(22), 9331-9337) and Prox-1 suppressors (U.S. Patent Publication No.2007/0026405).

In some embodiments, high throughput screening methods involve providinga combinatorial chemical or peptide library containing a large number ofpotential therapeutic compounds (potential modulator compounds). Such“combinatorial chemical libraries” or “ligand libraries” are thenscreened in one or more assays, as described herein, to identify thoselibrary members (particular chemical species or subclasses) that displaya desired characteristic activity. The compounds thus identified canserve as conventional “lead compounds” or can themselves be used aspotential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g. Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), andsmall organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

In other embodiments, agents that modulate 15-PGDH levels includeantisense molecules, ribozymes, DNAzymes, small inhibitory RNAs and thelike that act on other proteins, the silencing of which causes aninduction in 15-PGDH levels (e.g., silencing of beta-catenintranscription factor).

Methods of Screening for Modulators of 15-PGDH

A number of different screening protocols can be utilized to identifyagents that modulate the level of expression or activity of 15-PGDH incells, particularly mammalian cells, and especially human cells. Ingeneral terms, the screening methods involve screening a plurality ofagents to identify an agent that modulates the activity of 15-PGDH by,e.g., binding to a 15-PGDH polypeptide, preventing an inhibitor oractivator from binding to 15-PGDH, increasing association of aninhibitor or activator with 15-PGDH, or activating expression of15-PGDH.

Any cell expressing 15-PGDH or a fragment thereof can be used toidentify modulators. In some embodiments, the cells are eukaryotic cellslines (e.g., CHO or HEK293) transformed to express a heterologous15-PGDH polypeptide. In some embodiments, a cell expressing anendogenous 15-PGDH is used in screens. In other embodiments, modulatorsare screened for their ability to effect the levels of PGE₂.

Binding Assays

Preliminary screens can be conducted by screening for agents capable ofbinding to 15-PGDH, as at least some of the agents so identified arelikely 15-PGDH modulators. Binding assays are also useful, e.g., foridentifying endogenous proteins that interact with 15-PGDH. For example,antibodies, receptors or other molecules that bind 15-PGDH can beidentified in binding assays.

Binding assays usually involve contacting a 15-PGDH protein with one ormore test agents and allowing sufficient time for the protein and testagents to form a binding complex. Any binding complexes formed can bedetected using any of a number of established analytical techniques.Protein binding assays include, but are not limited to, methods thatmeasure co-precipitation or co-migration on non-denaturingSDS-polyacrylamide gels, and co-migration on Western blots (see, e.g.,Bennet, J. P. and Yamamura, H. I. (1985) “Neurotransmitter, Hormone orDrug Receptor Binding Methods,” in Neurotransmitter Receptor Binding(Yamamura, H. I., et al., eds.), pp. 61-89. Other binding assays involvethe use of mass spectrometry or NMR techniques to identify moleculesbound to 15-PGDH or displacement of labeled substrates. The 15-PGDHproteins utilized in such assays can be naturally expressed, cloned orsynthesized.

In addition, mammalian or yeast two-hybrid approaches (see, e.g.,Bartel, P. L. et. al. Methods Enzymol, 254:241 (1995)) can be used toidentify polypeptides or other molecules that interact or bind whenexpressed together in a host cell.

Expression Assays

Screening for a compound that modulates the expression of 15-PGDH arealso provided. Screening methods generally involve conducting cell-basedassays in which test compounds are contacted with one or more cellsexpressing 15-PGDH, and then detecting an increase or decrease in15-PGDH expression (either transcript or translation product). Assayscan be performed with any cells that express 15-PGDH.

15-PGDH expression can be detected in a number of different ways. Asdescribed infra, the expression level of 15-PGDH in a cell can bedetermined by probing the mRNA expressed in a cell with a probe thatspecifically hybridizes with a transcript (or complementary nucleic acidderived therefrom) of 15-PGDH. Probing can be conducted by lysing thecells and conducting Northern blots or without lysing the cells using insitu-hybridization techniques. Alternatively, 15-PGDH protein can bedetected using immunological methods in which a cell lysate is probedwith antibodies that specifically bind to 15-PGDH.

Other cell-based assays involve reporter assays conducted with cellsusing standard reporter gene assays. These assays can be performed ineither cells that do, or do not, express 15-PGDH. Some of these assaysare conducted with a heterologous nucleic acid construct that includes a15-PGDH promoter that is operably linked to a reporter gene that encodesa detectable product. A number of different reporter genes can beutilized. Some reporters are inherently detectable. An example of such areporter is green fluorescent protein that emits fluorescence that canbe detected with a fluorescence detector, or a ranilla luciferase (seeFIG. 4 and Example 4). Other reporters generate a detectable product.Often such reporters are enzymes. Exemplary enzyme reporters include,but are not limited to, β-glucuronidase, CAT (chloramphenicol acetyltransferase; Alton and Vapnek (1979) Nature 282:864-869), luciferase,β-galactosidase and alkaline phosphatase (Toh, et al. (1980) Eur. J.Biochem. 182:231-238; and Hall et al. (1983) J. Mol. Appl. Gen. 2: 101).

In these assays, cells harboring the reporter construct are contactedwith a test compound. Modulated promoter expression is monitored bydetecting the level of a detectable reporter. A number of differentkinds of 15-PGDH modulators can be identified in this assay. Forexample, a test compound that inhibits the promoter by binding to it,inhibits the promoter by binding to transcription factors or otherregulatory factors, binds to their promoter or triggers a cascade thatproduces a molecule that inhibits the promoter can be identified.Similarly a test compound that, e.g., activates the promoter by bindingto it, activates the promoter by binding to transcription factors orother regulatory factors, binds to their promoter or triggers a cascadethat produces a molecule that activates the promoter can also beidentified.

The level of expression or activity can be compared to a baseline value.The baseline value can be a value for a control sample or a statisticalvalue that is representative of 15-PGDH expression levels for a controlpopulation (e.g., healthy individuals as described herein) or cells(e.g., tissue culture cells not exposed to a 15-PGDH modulator).Expression levels can also be determined for cells that do not express15-PGDH as a negative control.

A variety of different types of cells can be utilized in the reporterassays. Cells that express an endogenous 15-PGDH include, e.g.,monocytes, neutrophils, leukocytes or brain, spleen cells, skeletalmuscle or adipocytes. Cells that do not endogenously express 15-PGDH canbe prokaryotic, but are preferably eukaryotic. The eukaryotic cells canbe any of the cells typically utilized in generating cells that harborrecombinant nucleic acid constructs. Exemplary eukaryotic cells include,but are not limited to, yeast, and various higher eukaryotic cells suchas the HEK293, HepG2, COS, CHO and HeLa cell lines, as well asestablished cancer cell lines, e.g., colon cancer cell lines.

Various controls can be conducted to ensure that an observed activity isauthentic including running parallel reactions with cells that lack thereporter construct or by not contacting a cell harboring the reporterconstruct with test compound. Compounds can also be further validated asdescribed below.

In a preferred embodiment proteins described herein are used in drugscreening assays. The proteins, antibodies, nucleic acids, modifiedproteins and cells are used in drug screening assays or by evaluatingthe effect of drug candidates on a “gene expression profile” orexpression profile of polypeptides. In a preferred embodiment, theexpression profiles are used, preferably in conjunction with highthroughput screening techniques to allow monitoring for expressionprofile genes after treatment with a candidate agent (e.g., Zlokarnik,et al., Science 279:84-8 (1998); Heid, Genome Res 6:986-94, 1996).

In a preferred embodiment, the proteins, antibodies, nucleic acids,modified proteins and cells containing the native or modified proteinsare used in screening assays. That is, the present invention providesmethods for screening for compositions which modulate the protein ormRNA levels of 15-PGDH or an identified physiological function of the15-PGDH protein. This can be done on an individual gene level or byevaluating the effect of drug candidates on a “gene expression profile”.In a preferred embodiment, the expression profiles are used, preferablyin conjunction with high throughput screening techniques to allowmonitoring for expression profile genes after treatment with a candidateagent, see Zlokarnik, supra.

Expression monitoring can be performed to identify compounds that modifythe expression of 15-PGDH. Generally, in a preferred embodiment, a testcompound is added to the cells prior to analysis. Moreover, screens arealso provided to identify agents that modulate the growth of or preventcolorectal cancer, modulate 15-PGDH, bind to 15-PGDH, or interfere withthe binding of a 15-PGDH protein and an antibody, substrate, or otherbinding partner.

Compositions, Kits and Integrated Systems

The invention provides compositions, kits and integrated systems forpracticing the assays described herein using nucleic acids encoding the15-PGDH polypeptides of the invention, or 15-PGDH proteins, anti-15-PGDHantibodies, etc.

The invention provides assay compositions for use in solid phase assays;such compositions can include, for example, one or more nucleic acidsencoding a 15-PGDH immobilized on a solid support, and a labelingreagent. In each case, the assay compositions can also includeadditional reagents that are desirable for hybridization. Modulators ofexpression or activity of a 15-PGDH of the invention can also beincluded in the assay compositions.

The invention also provides kits for carrying out the assays of theinvention. The kits typically include a probe that comprises an antibodythat specifically binds to 15-PGDH or a polynucleotide sequence encodinga 15-PGDH polypeptide, and a label for detecting the presence of theprobe. The kits may include at least one polynucleotide sequenceencoding a 15-PGDH polypeptides of the invention. Kits can include anyof the compositions noted above, and optionally further includeadditional components such as instructions to practice a high-throughputmethod of assaying for an effect on expression of the genes encoding the15-PGDH polypeptides of the invention, or on activity of the 15-PGDHpolypeptides of the invention, one or more containers or compartments(e.g., to hold the probe, labels, or the like), a control modulator ofthe expression or activity of 15-PGDH polypeptides, a robotic armaturefor mixing kit components or the like.

The invention also provides integrated systems for high-throughputscreening of potential modulators for an effect on the expression oractivity of the 15-PGDH polypeptides of the invention. The systems caninclude a robotic armature which transfers fluid from a source to adestination, a controller which controls the robotic armature, a labeldetector, a data storage unit which records label detection, and anassay component such as a microtiter dish comprising a well having areaction mixture or a substrate comprising a fixed nucleic acid orimmobilization moiety.

A number of robotic fluid transfer systems are available, or can easilybe made from existing components. For example, a Zymate XP (ZymarkCorporation; Hopkinton, Mass.) automated robot using a Microlab 2200(Hamilton; Reno, Nev.) pipetting station can be used to transferparallel samples to 96 well microtiter plates to set up several parallelsimultaneous binding assays.

Optical images viewed (and, optionally, recorded) by a camera or anotherrecording device (e.g., a photodiode and data storage device) areoptionally further processed in any of the embodiments herein, e.g., bydigitizing the image and storing and analyzing the image on a computer.A variety of commercially available peripheral equipment and software isavailable for digitizing, storing and analyzing a digitized video ordigitized optical image, e.g., using PC (Intel x86 or Pentiumchip-compatible DOS®, OS2® WINDOWS®, WINDOWS NT®, WINDOWS95®,WINDOWS98®, or WINDOWS2000′ based computers), MACINTOSH®, or UNIX® based(e.g., SUN® work station) computers.

One conventional system carries light from the specimen field to acooled charge-coupled device (CCD) camera, in common use in the art. ACCD camera includes an array of picture elements (pixels). The lightfrom the specimen is imaged on the CCD. Particular pixels correspondingto regions of the specimen (e.g., individual hybridization sites on anarray of biological polymers) are sampled to obtain light intensityreadings for each position. Multiple pixels are processed in parallel toincrease speed. The apparatus and methods of the invention are easilyused for viewing any sample, e.g., by fluorescent or dark fieldmicroscopic techniques.

Administration and Pharmaceutical Compositions

Modulators of 15-PGDH can be administered directly to the mammaliansubject for modulation of 15-PGDH levels and/or activity in vivo.Administration is by any of the routes normally used for introducing amodulator compound into ultimate contact with the tissue to be treatedand is well known to those of skill in the art. Although more than oneroute can be used to administer a particular composition, a particularroute can often provide a more immediate and more effective reactionthan another route.

The pharmaceutical compositions of the invention may comprise apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers are determined in part by the particular composition beingadministered, as well as by the particular method used to administer thecomposition. Accordingly, there are a wide variety of suitableformulations of pharmaceutical compositions of the present invention(see, e.g., Remington's Pharmaceutical Sciences, 17^(th) ed. 1985)).

The modulators (e.g., agonists or antagonists) of the expression oractivity of the 15-PGDH, alone or in combination with other suitablecomponents (e.g., an NSAID), can be made into aerosol formulations(i.e., they can be “nebulized”) to be administered via inhalation.Aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike.

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, orally, nasally, topically, intravenously,intraperitoneally, or intrathecally. The formulations of compounds canbe presented in unit-dose or multi-dose sealed containers, such asampoules and vials. Solutions and suspensions can be prepared fromsterile powders, granules, and tablets of the kind previously described.The modulators can also be administered as part of a prepared food ordrug.

The dose administered to a patient, in the context of the presentinvention should be sufficient to induce a beneficial response in thesubject over time. The optimal dose level for any patient will depend ona variety of factors including the efficacy of the specific modulatoremployed, the age, body weight, physical activity, and diet of thepatient, on a possible combination with other drugs, and on the severityof the case of diabetes. It is recommended that the daily dosage of themodulator be determined for each individual patient by those skilled inthe art in a similar way as for known insulin compositions. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects that accompany the administration of aparticular compound or vector in a particular subject.

In determining the effective amount of the modulator to be administereda physician may evaluate circulating plasma levels of the modulator,modulator toxicity, and the production of anti-modulator antibodies. Ingeneral, the dose equivalent of a modulator is from about 1 ng/kg to 10mg/kg for a typical subject.

For administration, 15-PGDH modulators of the present invention can beadministered at a rate determined by the LD-50 of the modulator, and theside-effects of the modulator at various concentrations, as applied tothe mass and overall health of the subject. Administration can beaccomplished via single or divided doses.

The compounds of the present invention can also be used effectively incombination with one or more additional active agents (e.g., an NSAID)depending on the desired target therapy. Combination therapy includesadministration of a single pharmaceutical dosage formulation thatcontains a 15-PGDH modulator of the invention and one or more additionalactive agents, as well as administration of a 15-PGDH modulator and eachactive agent in its own separate pharmaceutical dosage formulation. Forexample, a 15-PGDH modulator and celecoxib can be administered to thehuman subject together in a single oral dosage composition, such as atablet or capsule, or each agent can be administered in separate oraldosage formulations. In other embodiments, an NSAID, e.g., celecoxib oraspirin, may be administered with an effective amount of the 15-PGDHprotein. Where separate dosage formulations are used, an 15-PGDHmodulator and one or more additional active agents can be administeredat essentially the same time (i.e., concurrently), or at separatelystaggered times (i.e., sequentially). Combination therapy is understoodto include all these regimens.

Gene Therapy

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered polypeptides of 15-PGDHin mammalian cells or target tissues. Such methods can be used toadminister nucleic acids encoding polypeptides of the invention (e.g.,15-PGDH, including variants thereof) to cells in vitro. In someembodiments, the nucleic acids encoding polypeptides of the inventionare administered for in vivo or ex vivo gene therapy uses. Non-viralvector delivery systems include DNA plasmids, naked nucleic acid, andnucleic acid complexed with a delivery vehicle such as a liposome. Viralvector delivery systems include DNA and RNA viruses, which have eitherepisomal or integrated genomes after delivery to the cell. For a reviewof gene therapy procedures, see Anderson, Science 256:808-813 (1992);Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988);Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer &Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada etal., in Current Topics in Microbiology and Immunology Doerfler and Böhm(eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994). Methods ofnon-viral delivery of nucleic acids encoding engineered polypeptides ofthe invention include lipofection, microinjection, biolistics,virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acidconjugates, naked DNA, artificial virions, and agent-enhanced uptake ofDNA. Lipofection is described in e.g., U.S. Pat. No. 5,049,386, U.S.Pat. No. 4,946,787, and U.S. Pat. No. 4,897,355 and lipofection reagentsare sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic andneutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Felgner, WO 91/17424, WO91/16024. Delivery can be to cells (ex vivo administration) or targettissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered polypeptides of the invention take advantageof highly evolved processes for targeting a virus to specific cells inthe body and trafficking the viral payload to the nucleus. Viral vectorscan be administered directly to patients (in vivo) or they can be usedto treat cells in vitro and the modified cells are administered topatients (ex vivo). Conventional viral based systems for the delivery ofpolypeptides of the invention could include retroviral, lentivirus,adenoviral, adeno-associated and herpes simplex virus vectors for genetransfer. Viral vectors are currently the most efficient and versatilemethod of gene transfer in target cells and tissues. Integration in thehost genome is possible with the retrovirus, lentivirus, andadeno-associated virus gene transfer methods, often resulting in longterm expression of the inserted transgene. Additionally, hightransduction efficiencies have been observed in many different celltypes and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immuno deficiency virus (SW), human immuno deficiency virus(HIV), and combinations thereof (see, e.g. Buchscher et al., J. Virol.66,2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications where transient expression of the polypeptides of theinvention is preferred, adenoviral based systems are typically used.Adenoviral based vectors are capable of very high transductionefficiency in many cell types and do not require cell division. Withsuch vectors, high titer and levels of expression have been obtained.This vector can be produced in large quantities in a relatively simplesystem. Adeno-associated virus (“AAV”) vectors are also used totransduce cells with target nucleic acids, e.g., in the in vitroproduction of nucleic acids and peptides, and for in vivo and ex vivogene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994)).Construction of recombinant AAV vectors are described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al.; Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

pLASN and MFG-S are examples are retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 by invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)).

Replication-deficient recombinant adenoviral vectors (Ad) can beengineered such that a transgene replaces the Ad E1a, E1b, and E3 genes;subsequently the replication defector vector is propagated in human 293cells that supply deleted gene function in trans. Ad vectors cantransduce multiply types of tissues in vivo, including nondividing,differentiated cells such as those found in the liver, kidney and musclesystem tissues. Conventional Ad vectors have a large carrying capacity.An example of the use of an Ad vector in a clinical trial involvedpolynucleotide therapy for antitumor immunization with intramuscularinjection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Additionalexamples of the use of adenovirus vectors for gene transfer in clinicaltrials include Rosenecker et al., Infection 24:15-10 (1996); Sterman etal., Hum. Gene Ther. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Then.2:205-18 (1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topfet al., Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther.7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and 42 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by producer cell linethat packages a nucleic acid vector into a viral particle. The vectorstypically contain the minimal viral sequences required for packaging andsubsequent integration into a host, other viral sequences being replacedby an expression cassette for the protein to be expressed. The missingviral functions are supplied in trans by the packaging cell line. Forexample, AAV vectors used in gene therapy typically only possess ITRsequences from the AAV genome which are required for packaging andintegration into the host genome. Viral DNA is packaged in a cell line,which contains a helper plasmid encoding the other AAV genes, namely repand cap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. A viral vector is typically modified to have specificityfor a given cell type by expressing a ligand as a fusion protein with aviral coat protein on the viruses outer surface. The ligand is chosen tohave affinity for a receptor known to be present on the cell type ofinterest. For example, Han et al., PNAS 92:9747-9751 (1995), reportedthat Moloney murine leukemia virus can be modified to express humanheregulin fused to gp70, and the recombinant virus infects certain humanbreast cancer cells expressing human epidermal growth factor receptor.This principle can be extended to other pairs of virus expressing aligand fusion protein and target cell expressing a receptor. Forexample, filamentous phage can be engineered to display antibodyfragments (e.g., FAB or Fv) having specific binding affinity forvirtually any chosen cellular receptor., Although the above descriptionapplies primarily to viral vectors, the same principles can be appliedto nonviral vectors. Such vectors can be engineered to contain specificuptake sequences thought to favor uptake by specific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a nucleicacid (gene or cDNA) encoding a polypeptides of the invention, andre-infused back into the subject organism (e.g., patient). Various celltypes suitable for ex vivo transfection are well known to those of skillin the art (see, e.g., Freshney et al., Culture of Animal Cells, AManual of Basic Technique (3rd ed. 1994)) and the references citedtherein for a discussion of how to isolate and culture cells frompatients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med.176:1693-1702 (1992)).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+ (T cells), CD45+ (panB cells), GR-1(granulocytes), and Iad (differentiated antigen presenting cells) (seeInaba et al., J. Exp. Med. 176:1693-1702 (1992)).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic nucleic acids can be also administered directly to theorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells. Suitable methods of administering such nucleic acids areavailable and well known to those of skill in the art, and, althoughmore than one route can be used to administer a particular composition,a particular route can often provide a more immediate and more effectivereaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention, as described below (see, e.g., Remington'sPharmaceutical Sciences, 17th ed., 1989).

15-PGDH Nucleic Acid and Their Uses

Nucleic acids disclosed herein may be used in several ways. In a firstembodiment, nucleic acid probes are made and attached to biochips to beused in screening and diagnostic methods, as outlined below, or foradministration, e.g., for gene therapy, and/or antisense applications.Alternatively, nucleic acids that include coding regions of 15-PGDH canbe put into expression vectors for the expression of the proteins, againfor screening purposes or for administration to a subject. Two isoformsof 15-PGDH are known: 1) NM_(—)000860.4 corresponding to NP_(—)000851.2(isoform 1) and 2) NM_(—)001145816.1 corresponding to NP_(—)001139288.1(isoform 2). Isoform 2 lacks exon 6 and, therefore, corresponds to acatalytically dead variant. For any of the diagnostic and screeningmethods described herein, the levels of either isoform can be detectedand/or measured.

In a preferred embodiment, nucleic acid probes to a nucleic aciddisclosed herein (both the nucleic acid sequences outlined in thefigures and/or the complements thereof) are made. The nucleic acidprobes attached to the biochip are designed to be substantiallycomplementary to 15-PGDH nucleic acids, i.e. the target sequence (eitherthe target sequence of the sample or to other probe sequences, e.g., insandwich assays), such that hybridization of the target sequence and theprobes of the present invention occurs. As outlined below, thiscomplementarity need not be perfect; there may be any number of basepair mismatches which will interfere with hybridization between thetarget sequence and the single stranded nucleic acids of the presentinvention. However, if the number of mutations is so great that nohybridization can occur under even the least stringent of hybridizationconditions, the sequence is not a complementary target sequence. Thus,by “substantially complementary” herein is meant that the probes aresufficiently complementary to the target sequences to hybridize underappropriate reaction conditions, particularly high stringencyconditions, as outlined herein.

A nucleic acid probe is generally single stranded but can be partiallysingle and partially double stranded. The strandedness of the probe isdictated by the structure, composition, and properties of the targetsequence. In general, the nucleic acid probes range from about 8 toabout 100 bases long, with from about 10 to about 80 bases beingpreferred, and from about 30 to about 50 bases being particularlypreferred. That is, generally complements of ORFs or whole genes are notused. In some embodiments, nucleic acids of lengths up to hundreds ofbases can be used.

In a preferred embodiment, more than one probe per sequence is used,with either overlapping probes or probes to different sections of thetarget being used. That is, two, three, four or more probes, with threebeing preferred, are used to build in a redundancy for a particulartarget. The probes can be overlapping (i.e., have some sequence incommon), or separate. In some cases, PCR primers may be used to amplifysignal for higher sensitivity.

15-PGDH nucleic acids may be used to make a variety of expressionvectors to express proteins which can then be used in screening assaysor to generate antibodies, as described herein. Expression vectors andrecombinant DNA technology are well known to those of skill in the art(see, e.g., Ausubel, supra, and Gene Expression Systems (Fernandez &Hoeffler, eds, 1999)) and are used to express proteins. The expressionvectors may be either self-replicating extrachromosomal vectors orvectors which integrate into a host genome. Generally, these expressionvectors include transcriptional and translational regulatory nucleicacid operably linked to the nucleic acid encoding the metastaticcolorectal cancer protein.

The term “control sequences” refers to DNA sequences used for theexpression of an operably linked coding sequence in a particular hostorganism. Control sequences that are suitable for prokaryotes, e.g.,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers. Transcriptional andtranslational regulatory nucleic acid will generally be appropriate tothe host cell used to express the protein. Numerous types of appropriateexpression vectors, and suitable regulatory sequences are known in theart for a variety of host cells. In general, transcriptional andtranslational regulatory sequences may include, but are not limited to,promoter sequences, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, and enhancer oractivator sequences. In a preferred embodiment, the regulatory sequencesinclude a promoter and transcriptional start and stop sequences.Promoter sequences encode either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention. In addition, an expression vector may comprise additionalelements. For example, the expression vector may have two replicationsystems, thus allowing it to be maintained in two organisms, e.g., inmammalian or insect cells for expression and in a procaryotic host forcloning and amplification. Furthermore, for integrating expressionvectors, the expression vector contains at least one sequence homologousto the host cell genome, and preferably two homologous sequences whichflank the expression construct. The integrating vector may be directedto a specific locus in the host cell by selecting the appropriatehomologous sequence for inclusion in the vector. Constructs forintegrating vectors are well known in the art. In addition, in apreferred embodiment, the expression vector contains a selectable markergene to allow the selection of transformed host cells. Selection genesare well known in the art and will vary with the host cell used.

The 15-PGDH protein disclosed herein may be produced by culturing a hostcell transformed with an expression vector containing a nucleic acidencoding 15-PGDH, or functional fragments/variants thereof, under theappropriate conditions to induce or cause expression of the metastaticcolorectal cancer protein. Conditions appropriate for protein expressionwill vary with the choice of the expression vector and the host cell,and will be easily ascertained by one skilled in the art through routineexperimentation or optimization. For example, the use of constitutivepromoters in the expression vector will require optimizing the growthand proliferation of the host cell, while the use of an induciblepromoter requires the appropriate growth conditions for induction. Inaddition, in some embodiments, the timing of the harvest is important.For example, the baculoviral systems used in insect cell expression arelytic viruses, and thus harvest time selection can be crucial forproduct yield. Appropriate host cells include yeast, bacteria,archaebacteria, fungi, and insect and animal cells, including mammaliancells. Of particular interest are Saccharomyces cerevisiae and otheryeasts, E. coli, Bacillus subtilis, Sf9 cells, C129 cells, 293 cells,Neurospora, BHK, CHO, COS, HeLa cells, HLVEC (human umbilical veinendothelial cells), THP1 cells (a macrophage cell line) and variousother human cells and cell lines.

In one embodiment, the proteins are derivative or variant 15-PGDHproteins as compared to the wild-type sequence. That is, as outlinedmore fully below, the derivative peptide will often contain at least oneamino acid substitution, deletion or insertion, with amino acidsubstitutions being particularly preferred. The amino acid substitution,insertion or deletion may occur at a particular residue within thepeptide. Also included within one embodiment of 15-PGDH proteins of thepresent invention are amino acid sequence variants. These variantstypically fall into one or more of three classes: substitutional,insertional or deletional variants. These variants ordinarily areprepared by site specific mutagenesis of nucleotides in the encodingDNA, using cassette or PCR mutagenesis or other techniques, to produceDNA encoding the variant, and thereafter expressing the DNA inrecombinant cell culture as outlined above. However, variant proteinfragments having up to about 100-250 residues may be prepared by invitro synthesis. Amino acid sequence variants are characterized by thepredetermined nature of the variation, a feature that sets them apartfrom naturally occurring allelic or interspecies variation of themetastatic colorectal cancer protein amino acid sequence. The variantstypically exhibit the same qualitative biological activity as thenaturally occurring analogue, although variants can also be selectedwhich have modified characteristics as will be more fully outlinedbelow.

While the site or region for introducing an amino acid sequencevariation is often predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed variants screened for theoptimal combination of desired activity. Techniques exist for makingsubstitution mutations at predetermined sites in DNA having a knownsequence, e.g., M13 primer mutagenesis and PCR mutagenesis. Screening ofthe mutants is done using assays of protein activities.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be occasionally tolerated. Deletionsrange from about 1 to about 20 residues, although in some casesdeletions may be much larger.

Substitutions, deletions, truncations, insertions or any combinationthereof may be used to arrive at a final derivative. Generally thesechanges are done on a few amino acids to minimize the alteration of themolecule. Larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of a protein are desired,substitutions are generally made in accordance with the amino acidsubstitution commonly known in the art.

Variants typically exhibit the same qualitative biological activity andwill elicit the same immune response as the naturally-occurring analog,although variants also are selected to modify the characteristics of themetastatic colorectal cancer proteins as needed. Alternatively, thevariant may be designed or reorganized such that the biological activityof the is altered. For example, glycosylation sites may be altered orremoved. When a 15-PGDH protein is to be used to generate antibodies,e.g., for immunotherapy or immunodiagnosis, the protein should share atleast one epitope or determinant with the full length protein. By“epitope” or “determinant” herein is typically meant a portion of aprotein which will generate and/or bind an antibody or T-cell receptorin the context of MHC. Thus, in most instances, antibodies made to asmaller metastatic colorectal cancer protein will be able to bind to thefull-length protein, particularly linear epitopes. In a preferredembodiment, the epitope is unique; that is, antibodies generated to aunique epitope show little or no cross-reactivity. Methods of preparingpolyclonal or monoclonal antibodies are well known in the art (e.g.,Coligan, supra; and Harlow & Lane, supra; Kohler & Milstein, Nature256:495 (1975)).

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.Although exemplification is provided in the context of colon cancer, oneof skill in the art would readily appreciate the applicability of theinvention to other forms of cancer.

EXAMPLE 1 15-PGDH is required for celecoxib prevention of murine colontumors

To conduct this study FVB mice were selected, chosen because at baselinethis mouse strain is sensitive to AOM induced colon tumors (Nambiar, P.R. et al., (2003) Int J Oncol 22, 145-50). In 15-PGDH wild-type FVBmice, AOM induced 2.3±0.4 tumors per mouse colon (FIG. 1A, 1C), which onhistology review were all adenomatous lesions. As in human trials,administering dietary celecoxib protected wild-type FVB mice againstcolon tumor development, reducing adenoma development to 0.7±0.3 tumorsper mouse (P=0.003) (FIG. 1A, 1C). Furthermore, while nearly all tumorsarising in control mice exceeded 1 mm in size, tumors in celecoxibtreated mice rarely reached this size (2.2±0.4 versus 0.3±0.1, P<0.0001)(FIG. 1B).

Dietary celecoxib could thus near completely protect wild-type FVB micefrom developing colon tumors. Further investigation however revealedthat the ability of celecoxib to protect mice from colon tumors wascrucially dependent upon the concomitant activity of 15-PGDH, and thatthis protection could be abrogated by breeding 15-PGDH knockout allelesinto the FVB strain. Thus, celecoxib treated 15-PGDH null mice developed5.5-fold more colon adenomas than did their celecoxib treated 15-PGDHwild-type littermates (3.9±0.8 versus 0.7±0.3, P=0.0001) (FIG. 1A, 1C).Moreover, all colonic adenomas arising in celecoxib treated knockoutmice were large, each exceeding 1 mm in size; whereas, large tumors werenearly completely absent in celecoxib treated 15-PGDH wild-type mice(3.9±0.8 large tumors per knockout mouse versus 0.3±0.1 per wild-type,P=0.0001) (FIG. 1B, 1C). By several measures, 15-PGDH knockout mice werealmost completely resistant to the colon tumor prevention effects ofcelecoxib. For example, tumor development in the knockout mice did notsignificantly differ between celecoxib treated versus untreated animals(3.9±0.8 versus 4.9±0.8,respectively, P=0.36) (FIG. 1A). Moreover, colontumor development in celecoxib treated 15-PGDH knockout mice actuallyexceeded that of drug untreated wild-type mice (3.9±0.8 versus 2.2±0.4respectively, for tumors >1 mm in size, P=0.04; and, 3.9±0.8 versus2.3±0.4 respectively tumors of any size, P=0.06) (FIG. 1A, 1B). Weconclude that celecoxib mediated colon tumor prevention requiresconcurrent presence of the prostaglandin inactivating activity ofcolonic 15-PGDH, and that 15-PGDH loss confers in vivo resistance tothis drug effect.

EXAMPLE 2 15-PGDH is required for celecoxib reduction of colonic PGE₂

To further investigate the mechanism of celecoxib's dependence on15-PGDH, we determined PGE₂ levels in colonic mucosa of FVB mice underdifferent experimental conditions. Consistent with the role of 15-PGDHin mediating prostaglandin degradation, 15-PGDH gene knockoutessentially doubled FVB colonic PGE₂ levels (9.1±1.5 ng/mg protein inknockouts versus 5.70±0.8 ng/mg protein in controls, P=0.04) (FIG. 1D).Celecoxib treatment of 15-PGDH wild-type mice markedly lowered PGE₂levels to 1.6±0.4ng/mg protein (P<0.001) (FIG. 1D). In contrast, in15-PGDH knockout mice the biochemical activity of celecoxib was muchattenuated, with the drug lowering PGE₂ levels to only 4.7±0.8 ng/mgprotein. This level was three-fold the level achieved in drug treatedwild-type mice (P=0.0002), and was not significantly different from thePGE₂ level of drug free wild-type mice (P=0.4) (FIG. 1D). In overview,the efficacy of celecoxib in lowering colonic PGE₂ levels in thesedifferent models closely paralleled the drug's anti-tumor activity (FIG.1D versus FIG. 1A, 1B), and mice that lacked 15-PGDH equally acquiredresistance to celecoxib's biochemical activity of lowering colonic PGE₂and to celecoxib's phenotypic effect of preventing colon tumors.Celecoxib resistance of 15-PGDH null mice was not due to any change indrug absorption or catabolism, as tissue levels of celecoxib wereindistinguishable between drug treated 15-PGDH wild-type and null mice(39.5±9.7 versus 37.7±7.6 ng/mg protein, P=0.7) (FIG. 2A). Additionally,celecoxib did not regulate 15-PGDH levels, as Western blot analysisshowed equal colonic 15-PGDH amounts in celecoxib treated versusuntreated wild-type mice (FIG. 2B). Rather, the effective depletion ofcolonic PGE₂ required the COX-2 inhibitor drug to act in concert withthe independent prostaglandin degrading activity of 15-PGDH.

EXAMPLE 3 Low 15-PGDH levels are associated with celecoxib resistance inhumans with recurrent colon adenomas

These observations in mice suggest that humans with lower levels ofcolonic 15-PGDH might also be resistant to the colon tumor preventionactivity of celecoxib. To test this hypothesis, we examined frozenbiopsies of rectal mucosa obtained from 16 individuals at the time oftheir enrollment in the Adenoma Prevention with Celecoxib (APC) trial(Bertagnolli, M. M. et al., (2006) N Engl J Med 355, 873-84). Theseindividuals were all at high risk for colon adenoma development based onhaving had multiple colon adenomas and/or adenomas greater than 5 mm indiameter prior to their enrollment. Following clearing of their colonicadenomas by colonoscopy, each individual received 36-months of dailytreatment with celecoxib (Bertagnolli, M. M. et al., (2006) N Engl J Med355, 873-84). Measurement by real-time PCR of 15-PGDH transcript levelsin these pre-treatment biopsy samples showed a 12-fold variation fromlowest to highest 15-PGDH mRNA level among these 16 unrelatedindividuals (median=56.4, mean=66.2, range: 13.3-155.7) (FIG. 3). Repeatcolonoscopy after 36 months revealed that four of these individuals hadproven resistant to celecoxib treatment, as evidenced by the developmentof new adenomas, that all arose at new locations (FIG. 3). In total 9new adenomas were detected in these patients (FIG. 3). 15-PGDH levelsamong individuals who developed new adenomas were lower than 15-PGDHlevels among individuals who remained adenoma free (P=0.04) (FIG. 3).This can be further appreciated by noting that all four individuals withnew adenomas demonstrated 15-PGDH levels below the cohort mean (P=0.03)(FIG. 3). The relationship of low 15-PGDH level to celecoxib resistancebecomes even stronger if analyzed in terms of the numbers of newadenomas that individuals developed, with 8 of the 9 adenomatous polypsthat recurred during celecoxib treatment arising in individuals withcolonic 15-PGDH values below the cohort median (P=0.01), and with all 9new adenomas arising in individuals with colonic 15-PGDH below thecohort mean (P=0.001) (FIG. 3).

Accordingly, 15-PGDH activity can determine sensitivity or resistance tothe colon tumor preventive activity of celecoxib. Gene knock-out of15-PGDH confers near complete resistance to celecoxib colon tumorprevention in mice. More significantly, low levels of colonic 15-PGDHare associated with failure of celecoxib colon tumor prevention in man.These findings elucidate a previously unsuspected pharmacogeneticinteraction that bears on the differences among different individuals inthe efficacy of celecoxib treatment for prevention of colorectaladenomas. These observations show that measurement of 15-PGDH isclinically useful in selecting patients most likely to benefit fromtreatment with COX-2 inhibitors for colon tumor chemoprevention.Further, these observations also demonstrate the utility in identifyinggenetic markers to predict for individuals who are low expressers of15-PGDH. In addition, these new observations further document the roleof 15-PGDH as a key suppressor of tumor development in the colon. Theseresults also show that agents capable of inducing or reactivating15-PGDH expression provides new approaches for colon adenoma and cancerprevention, as well as colon cancer therapy.

Materials And Methods

Mouse breeding

Mouse studies were conducted in the Case Animal Resource Center under aprotocol approved by the Institutional Animal Care and Use Committee.15-Hydroxyprostaglandin dehydrogenase (15-PGDH) knockout mice on aC57BL/6J background were generated as described previously (Myung, S. J.et al., (2006) Proc Natl Acad Sci USA 103, 12098-102; Coggins, K. G. etal, (2002) Nat Med 8, 91-2) and were bred to generation F8 onto an FVB/N(Jackson Laboratory, Bar Harbor, Me.) background, with genotyping doneas previously described (Myung, S. J. et al., (2006) Proc Natl Acad SciU S A 103, 12098-102). 15-PGDH +/− mice generation F8 were intercrossedand siblings of 15-PGDH +/+ and −/− genotypes were selected out forstudies with AOM and celecoxib. Eight- to 12-week-old mice wereadministered AOM by i.p. injection once weekly for 6 weeks at 10mg/kgdose (Sigma Chemical Co., St. Louis, Mo.). Mice were euthanized 24 weeksafter the last AOM injection. After euthanizing, the colons were openedlongitudinally, rinsed with ice-cold PBS, and examined under adissecting microscope to identify all tumors. Tumors were resected,fixed in 10% neutral buffered formalin, and paraffin-embedded forhistologic examination. All mice received an AIN-76A diet (HarlanTeklad, Madison, Wis.). The diet of celecoxib treated mice wassupplemented with 1250 mg/kg of this agent (LKT laboratories, St. Paul,Minn.) (Williams, C. S. et al., (2000) Cancer Res 60, 6045-51). In AOMstudies, celecoxib supplementation was initiated starting the day offirst AOM injection and continued throughout the lifetime of the mouse.In studies of colonic prostaglandin levels celecoxib was administered toa cohort of 8-12 week old mice continuously for 2 weeks, at which timemice were euthanized.

PGE₂ analyses

Following 2 weeks of control or celecoxib supplemented diet mice wereeuthanized. The colons were opened and washed with ice cold PBS, and thecolon mucosa was then gently scraped, snap frozen in liquid nitrogen andstored at −80° C. PGE₂ analyses were performed as previously described(Myung, S. J. et al., (2006) Proc Natl Acad Sci U S A 103, 12098-102).To extract PGE₂ from mouse colon epithelial tissue, frozen mucosalsamples (25-50 mg) were ground to a fine powder in aliquid-nitrogen-cooled mortar (Fisher Scientific). Samples were thentransferred to sealed microcentrifuge tubes, and three times the volumeof ice-cold PBS buffer containing 0.1% butylated hydroxytoluene (BHT)and 1 mM EDTA were added. The sample was then homogenized in anultrasonic processor (Misonix, Farmingdale, N.J.) at 0° C. for 3 min. A100-μl aliquot of the homogenate was transferred to a glass tube (13×100mm) and subjected to extraction of eicosanoids by using a modifiedversion of the method of Kempen et al. (Kempen, E. C. et al., (2001)Anal Biochem 297, 183-90). Briefly, 20-μl aliquots of 1 N citric acidand 10 μl of deuterated PGE₂ (100 ng/ml) were added to the samples.Eicosanoids were then extracted with 1 ml of hexane:ethyl acetate (1:1,vol/vol) and vortex-mixed for 2 min. Samples were centrifuged at 1,800×gfor 10 min at 4° C. The upper organic layer was collected, and theorganic phases from three extractions were pooled and then evaporated todryness under a stream of nitrogen at room temperature. All extractionprocedures were performed at minimum light levels under cold conditions(4° C.). Samples were then reconstituted in 100 μl of methanol:ammoniumacetate buffer (10 mM, pH 8.5; 70:30, vol/vol) prior to liquidchromatography tandem MS (LC/MS/MS) analysis. The protein concentrationwas determined by a Bradford protein assay (Bio-Rad, Hercules, Calif.).PGE₂ was measured with reverse-phase LC electrospray ionization MS.LC/MS/MS analyses were performed with a Quattro Ultima tandem massspectrometer (Micromass, Beverly, Mass.) equipped with an Agilent HP1100 binary pump HPLC inlet. PGE₂ was separated by using a Luna 3pPhenyl-Hexyl 2×150 mm LC column (Phenomenex, Torrance, Calif.). Themobile phase consisted of 10 mM ammonium acetate (pH 8.5) and methanol.The flow rate was 250 μl/min with a column temperature of 50° C. Thesample injection volume was 25 μl. Samples were kept at 4° C. during theanalysis. The mass spectrometer was operated in the electrospraynegative-ion mode with a cone voltage of 100 V, a cone gas flow rate of117 liters/h, and a devolution gas flow rate of 998 liters/h. Thetemperature of the desolvation region was 400° C., and the temperatureof the source region was 120° C. Fragmentation for all compounds wasperformed by using argon as the collision gas at a collision cellpressure of 2.10×10⁻³ ton. The collision energy was 19 V. Prostaglandinswere detected by using electrospray negative ionization andmultiple-reaction monitoring of the transition ions for the PGE₂(351.2>271.2) and 13,14-dihydro-15-keto-PGE₂ (351.2>333.1). This methodproduces excellent linearity and a lower limit of quantitation of 10ng/ml, which is more than adequate to assess endogenous eicosanoidmetabolism in small (25- to 30-mg) amounts of tissue. The results wereexpressed as ng of eicosanoid per mg of protein.

Determination of celecoxib levels in mouse tissues by LC/MS/MS

Following 2 weeks of control or celecoxib supplemented diet mice wereeuthanized. The colons were opened and washed with ice cold PBS. Mucosafrom the distal 6 cm of mouse colon was then gently scraped, snap frozenin liquid nitrogen, and stored at −80 ° C. for analysis. Approximately10 mg of frozen tissue was ground to a fine powder using aliquid-nitrogen-cooled mortar (Fisher Scientific Co., Fair Lawn, N.J.).The samples were then transferred to microcentrifuge tubes and threevolumes of ice-cold PBS buffer were added before further homogenizationof the sample with ultrasonic tissues processor (Masonix). An aliquot(100 μl) of homogenate was transferred to a glass tube (13×100 mm). Tothe homogenate, 2 ml of hexane:ethyl acetate (1:1, v/v) was added; themixture was vortex mixed for 5 min. and then centrifuged at 4000 rpm at5° C. for 5 min. The extraction was repeated twice and the upper organiclayer was collected, pooled and evaporated to dryness under a stream ofnitrogen at room temperature. The sample was then reconstituted in 200μL of methanol: 10 mM ammonium acetate, pH 8.5 (1/1. v/v). The celecoxiblevel in the samples was determined by LC/MS/MS. The LC/MS/MS wasoperated under the same condition as described for measurement of PGE₂with minor changes. Briefly, ten microliters of the sample was injectedon a Luna 3 um phenyl-hexyl 2×150 mm analytical column (Phenomenex).Celecoxib was detected and quantified by operating the mass spectrometerin electrospray negative ion mode and monitoring the transition m/z380.2>316.1. Quantification was done by comparing the sample peak areasto a standard curve and concentration of celecoxib was normalized byprotein concentration. Preliminary studies in which celecoxib was spikedinto control normal mouse samples demonstrated that under the aboveconditions drug recovery consistently exceeded 95% over a wideconcentration range.

15-PGDH Western Blot analysis

Western assay of 15-PGDH was done as described previously, usingconditions in which the assay is in the linear range (Yan, M. et al.,(2004) Proc Natl Acad Sci U S A 101, 17468-73). Mucosa was collectedfrom the distal 6 cm of the colon by scraping, snap freezing in liquidnitrogen and storage at 80° C. Tissue lysates were prepared by pipettingin RIPA buffer (Upstate Biotechnology, Lake Placid, N.Y.) (50 mMTris.HCL/1% Nonidet P-40/0.25% Na-deoxycholate/150 mM NaCl/1 mM EDTA/1mM

PMSF) supplemented with protease inhibitor mixture (Roche, Indianapolis,Ind.), were separated on 12% SDS/PAGE Ready Gels (Pierce, Rockford,Ill.) (30 μg per lane), and transferred to Immobilon polyvinylidenedifluoride membrane (Millipore, Billerica, Mass.). The blots wereblocked with 5% milk, probed with monoclonal anti-PGDH antibody at a1:200 dilution (equaling 9.5n/m1) (Yan, M. et al., (2004) Proc Natl AcadSci USA 101, 17468-73) and with anti-actin antibody (Sigma-Aldrich, St.Louis, Mo.) at a 1:2000 dilution, developed by usinghorseradish-peroxidase-conjugated anti-mouse trueblot antibodies(eBiosciene, San Diego, Calif.), visualized by using an EnhancedChemiluminescence Plus detection kit (Amersham Biosciences, Piscataway,N.J.), following the manufacturer's instructions, and then scanned on aPhosphorImager (Molecular Dynamics, Calif.).

Human subjects

We performed an IRB approved prospective, randomized trial of the NSAID,celecoxib, for prevention of sporadic colorectal adenomas (Bertagnolli,M. M. et al., (2006) N Engl J Med 355, 873-84). In this study, known asthe Adenoma Prevention with Celecoxib (APC) Trial, 2035 patients with ahistory of colorectal adenomas were randomized to receive eitherplacebo, celecoxib 200 mg twice daily, or celecoxib 400 mg twice daily,and were followed for 36 months. Celecoxib at either dose significantlyreduced the incidence of adenomas detected at the end of the 36 monthsurveillance interval. A subset of patients on the APC trial alsounderwent a separate pre-treatment endoscopic procedure prior toinitiating celecoxib use, during which biopsies of normal rectal mucosawere obtained. Mucosal specimens (2mm diameter) were flash frozen inliquid nitrogen and stored at −80° C. until assay for 15-PGDH.

Human 15-PGDH transcript measurement

RNA was isolated from colon mucosal biopsies using an RNAqueous® kit(Ambion, Austin, Tex.) following the manufacturer's recommended protocolwith minor alterations. Briefly, approximately 10 mg of frozen tissuewas ground to a fine powder using a liquid-nitrogen-cooled mortar. Thesamples were then transferred to microcentrifuge tubes containing 400 μlof Lysis/Binding solution. Each sample was then passed through a26-gauge needle several times until no longer viscous and then loadedonto a filter cartridge for the subsequent washing steps. RNA from eachsample was eluted from the filter cartridge using 40 μl of ElutionSolution followed by another elution with 20 μl of Elution Solution, fora pooled elution volume of 60 μl. Concentration and quality of the RNAsamples were determined using a ND-1000 Spectrophotometer (NanoDrop,Wilmington, Del.) and 500 ng of sample was used for cDNA synthesis andsubsequent real-time PCR assays for 15-PGDH. cDNA was synthesized usingAMV Reverse Transcriptase (Roche, Indianapolis, Ind.) following themanufacturer's recommended protocol. Real-time PCR measurement of15-PGDH was performed using the human 15-PGDH Taqman Probe/Primer kitHs00168359_ml from Applied Biosystems (Foster City, CA) and 1× IQSupermix from Bio-Rad (Hercules, Calif.), and detected in an Icycler™optical module (Biorad, Hercules, Calif.) (Yan, M. et al., (2004) ProcNatl Acad Sci USA 101, 17468-73). A 25 ul reaction mix contained a 1:20dilution of primer/probe in 1× Supermix (Bio-Rad, CA). Thermal cyclingwas initiated at 95° C. for 3 min, followed by 50 cycles of 95° C. for15 sec and 60° C. for 1 min. Cytokeratin 20 (Krt20), a marker of colonicepithelial cell mass, was used as the endogenous control and wasamplified using the human Krt20 TaqMan primer/probe kit Hs00300643_mlfrom Applied Biosystems and 1× IQ Supermix, with PCR initiated at 95° C.for 10 min, followed by 50 cycles of 95° C. for 15 sec and 60° C. for 1min. The level of 15-PGDH

RNA was determined as the ratio of 15-PGDH:Krt20 =2 exp(CT_(PGDH)−CT_(Krt20)). Plotted values of 15-PGDH represent numericalaverages of 15-PGDH levels assayed from three independent reversetranscription reactions. Additionally, for each reverse transcriptionreaction, CT_(Krt20) and CT_(PGDH) were determined as the average valuesobtained from three independent real-time PCR reactions.

Statistical analyses

Prostaglandin and celecoxib levels were log transformed in order to beapproximately normally distributed for analysis with a generalizedlinear regression model with contrasts, generating two-sided P-values.Tumor numbers in AOM-treated mice were analyzed using a negativebinomial generalized linear model with contrasts, generating two-sidedP-values. Results of all mouse studies in the manuscript are presentedas mean values±standard errors of the mean. In humans, associationsbetween presence or absence of tumor relapse and 15-PGDH level (treatedas a continuous variable) was analyzed using a negative binomialgeneralized model with contrasts, generating a two-sided P-value(P=0.04). Comparison of median 15-PGDH levels in individuals whorelapsed versus those who did not relapse was also done using a Wilcoxonsigned-rank test, generating a one-sided P-value (P=0.04) for thepre-specified model that individuals with lower 15-PGDH levels would becelecoxib resistant. And, association between presence or absence ofrelapse in individuals and 15-PGDH dichotomized by the population meanwas done using a Poisson generalized linear model with contrasts,generating a two sided P-value (P=0.03). Association between numbers ofrecurrent adenoma tumors in celecoxib treated individuals and 15-PGDHlevels dichotomized either by the population median or the populationmean, was analyzed using a Poisson generalized linear model withcontrasts, generating two-sided P-values (respectively P=0.01 andP=0.001).

Incorporation by Reference

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference. In case of conflict, the present application, including anydefinitions herein, will control.

Equivalents

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

What is claimed is:
 1. A method of treating a non-steroidalanti-inflammatory drug (NSAID)-responsive condition in a human subject,wherein the NSAID-responsive condition is selected from the groupconsisting of: an inflammatory disorder, an immunologic disorder, andcolon neoplasia, and wherein the subject has been determined to haveincreased or decreased levels of 15-hydroxyprostaglandin dehydrogenase(15-PGDH), the method comprising: (i) administering to the subject aneffective amount of an NSAID if the levels of 15-PGDH are increased; or(ii) administering to the subject an effective amount of an NSAID and anagent that upregulates 15-PGDH, wherein the agent is not a Prox-1suppressor, if the levels of 15-PGDH are decreased.
 2. The methodaccording to claim 1, wherein the NSAID-responsive condition is colonneoplasia.
 3. The method according to claim 2, wherein the colonneoplasia is colon cancer.
 4. The method according to claim 2, whereinthe colon neoplasia is colon adenoma.
 5. The method according to claim1, wherein the NSAID-responsive condition is an inflammatory disorder oran immunologic disorder.
 6. The method according to claim 1, wherein theNSAID is celecoxib or aspirin.
 7. The method according to claim 1,wherein the agent is selected from the group consisting of: a smallmolecule, a polypeptide, nucleic acid, aptamers, and antibody.
 8. Themethod according to claim 7, wherein the small molecule is erlotinib orbutyrate.
 9. The method according to claim 7, wherein the nucleic acidis an siRNA or antisense.
 10. The method according to claim 9, whereinthe siRNA inhibits the expression of beta-catenin transcription factor.11. The method according to claim 1, wherein the agent directly orindirectly upregulates 15-PGDH levels or activity.
 12. A method ofdecreasing non-steroidal anti-inflammatory drug (NSAID) resistance in ahuman subject, wherein the subject has an NSAID-responsive conditionselected from the group consisting of: an inflammatory disorder, animmunologic disorder, and colon neoplasia, and wherein the subject hasbeen determined to have decreased levels of 15-hydroxyprostaglandindehydrogenase (15-PGDH), the method comprising administering to thesubject: (i) an effective amount of a compound that increases 15-PGDHlevels or 15-PGDH activity; or (ii) an effective amount of 15-PGDHprotein, cDNA, or an active fragment thereof, wherein a compound thatincreases 15-PGDH levels is not a Prox-1 suppressor.
 13. The methodaccording to claim 12, wherein the 15-PGDH levels include protein, mRNA,or cDNA level of 15-PGDH.
 14. The method according to claim 12, whereinthe compound is selected from the group consisting of: a small molecule,a polypeptide, a nucleic acid, an aptamer, and an antibody.
 15. Themethod according to claim 14, wherein the nucleic acid is an siRNA orantisense.
 16. The method according to claim 15, wherein the siRNAinhibits the expression of beta-catenin transcription factor.
 17. Themethod according to claim 12, wherein the compound directly orindirectly upregulates 15-PGDH levels or activity.
 18. A method oftreating a non-steroidal anti-inflammatory drug (NSAID)-responsivecondition in a human subject, wherein the NSAID-responsive condition iscolon neoplasia, and wherein the subject has been determined to havedecreased levels of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), themethod comprising administering to the subject an effective amount of anNSAID and an agent that upregulates 15-PGDH.
 19. The method of claim 18,wherein the agent is erlotinib or butyrate.
 20. A method of treating anon-steroidal anti-inflammatory drug (NSAID)-responsive condition in ahuman subject, wherein the NSAID-responsive condition is colonneoplasia, and wherein the subject has been determined to have increasedlevels of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the methodcomprising administering to the subject an effective amount of an NSAID.21. The method according to any of claims 18-20, wherein the NSAID iscelecoxib or aspirin.